Control of cellulose biosynthesis by overexpression of a transcription factor

ABSTRACT

The invention relates to nucleic acids, proteins and methods for modulating the cellulose content of plants.

This application is a continuation of U.S. patent application Ser. No.14/381,040, filed Aug. 26, 2014, which is a U.S. National StageApplication under 35 U.S.C. § 371 of PCT/US2013/027777 filed on Feb. 26,2013, and published on Sep. 6, 2013 as WO 2013/130456 A2, which claimsbenefit of the filing date of U.S. Provisional Application Ser. No.61/603,823, filed Feb. 27, 2012, the contents of which are specificallyincorporated herein by reference in their entirety.

This invention was made with government support under DE-FC02-07ER64494awarded by the U.S. Department of Energy. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Cellulose is a complex carbohydrate that serves as the basic structuralcomponent of plant cell walls. Cellulose accounts for roughly one thirdof all vegetal matter, making it the most common organic compound onearth. Due to its ubiquitous nature, cellulose and its derivatives arekey resources to many industries, such as agricultural, forestry,textile, and paper industries. Recently, there has also been a growinginterest in using cellulose to produce value-added compounds such asethanol or butanol (e.g., for use as biofuels). For industries that relyon plant biomass, for example, the timber and fiber industries,profitability is directly related to the quantity and quality ofcellulose harvested from crops. However, there are currently no knownmethods of genetically controlling the quantity or quality of cellulosesynthesized in plant species.

SUMMARY OF THE INVENTION

The invention relates to nucleic acids and proteins useful forregulating expression of plant genes. In some embodiments, theapplication relates to transgenic plants and compositions derivedtherefrom that have increased cellulose content, as well as to methodsof directly regulating cellulose biosynthesis through geneticmanipulation and control. As described herein, several transcriptionfactors directly activate the expression of cellulose synthases. Whenthese transcription factors activate the expression of cellulosesynthases the synthases produce increased percentages of cellulose. Thenucleic acids, proteins and methods described herein can therefore beused to increase the amount and quality of cellulose in plants. Suchregulation of plant cellulose quality and quantity can reduce the costsand improve the efficiencies of industries such as the paper, fiber, andlumber industries.

One aspect of the invention is a plant comprising an isolated nucleicacid encoding a plant transcription factor selected from the groupconsisting of MYB46, HAM1, HAM2, MYB112, WRKY11, ERF6, or a combinationthereof. In some embodiments, the plant transcription factor can, forexample, be MYB46. The isolated nucleic acid can have a heterologouspromoter segment operably linked to a nucleic segment that encodes theplant transcription factor coding region. Such a heterologous promoteris not the plant transcription factor's natural or native promoter. Forexample, the heterologous promoter can be a strong promoter, weakpromoter, inducible promoter, tissue specific promoter, developmentallyregulated promoter, or a combination of such promoters. The isolatednucleic acid can express increased levels of the plant transcriptionfactor in the plant compared to a corresponding transcription factorgene naturally present in a wild type plant of the same species. Theplant with the isolated nucleic acid encoding the plant transcriptionfactor can express increased levels of secondary wall cellulose comparedto a wild type plant of the same species without the isolated nucleicacid. For example, such a plant can have at least about 2% increasedcellulose content compared to a wild type plant of the same species thatdoes not have the isolated nucleic acid. The plant can be a transgenicplant, a genetically modified plant, or a plant selectively bred tocomprise the isolated nucleic acid.

Another aspect is a seed that includes an isolated nucleic acid encodinga plant transcription factor selected from the group consisting ofMYB46, HAM1, HAM2, MYB112, WRKY11, ERF6, or a combination thereof. Theisolated nucleic acid included within the seed can include aheterologous promoter segment operably linked to a nucleic segment thatencodes the plant transcription factor coding region. Such aheterologous promoter is not the plant transcription factor's naturalpromoter, but can be a strong, weak, inducible, tissue specific,developmentally regulated or a combination thereof.

Another aspect is a plant biomass that includes secondary wall celluloseisolated from a plant that includes an isolated nucleic acid encoding aplant transcription factor selected from the group consisting of MYB46,HAM1, HAM2, MYB112, WRKY11, ERF6, or a combination thereof.

A further aspect is a method of increasing cellulose content in a plantcell that includes transforming the plant cell with an isolated nucleicacid that can express a transcription factor selected from the groupconsisting of MYB46, HAM1, HAM2, MYB112, WRKY11, ERF6, and anycombination thereof. The isolated nucleic acid can include aheterologous promoter segment operably linked to a nucleic segment thatencodes the plant transcription factor coding region. For example, sucha heterologous promoter is not the plant transcription factor's naturalpromoter. Instead, the heterologous promoter can be a strong promoter,weak promoter, inducible promoter, tissue specific promoter,developmentally regulated promoter, or a combination thereof.

These and other aspects of the invention are further described herein.

DESCRIPTION OF THE DRAWINGS

FIG. 1A-1C show images of transgenic plants and bar graphs illustratingphenotypic effects of the transcription factor MYB46 on the expressionof the cellulose synthase genes CESA4, CESA7 and CESA8. FIG. 1A showsimages of wild type Arabidopsis (WT) and transgenic Arabidopsis plants.The transgenic Arabidopsis plants OX8 and OX9 over-express the MYB46transcription factor, while the DEX transgenic Arabidopsis plant has adexamethasone-inducible MYB46 transgene. MYB46 expression in the DEXplants was induced by 24 hr of dexamethasone treatment (+DEX). As acontrol, a DEN plant was mock-treated for 24 hr with 0.05% ethanol andof 0.02% silwet surfactant (−DEX). The plants shown are three-weeks old.As illustrated, the MYB46 over-expression, and DEX(+) plants exhibit aleaf curling phenotype. FIG. 1B is a bar graph illustrating relativeMYB46 expression levels. FIG. 1C is a bar graph illustrating relativeexpression levels of CESA4, CESA7 and CESA8 genes in transgenic OX8 andOX9 Arabidopsis plants that over-express the MYB46 transcription factor,as well as the DEX transgenic Arabidopsis plant having adexamethasone-inducible MYB46 transgene. Expression levels weredetermined by real-time PCR analysis. As shown, MYB46 up-regulates theexpression of CESA4, CESA7 and CESA8 genes. Error bars represent thestandard deviation of three biological replicates.

FIG. 2A-D illustrate that MYB46 directly activates the expression ofCESA4, CESA7 and CESA8. FIG. 2A is a diagram of the effector andreporter constructs used in some experiments. MYB46 was fused with anucleic acid encoding the glucocorticoid receptor (GR) and this MYB46-GRfusion was expressed via the CaMV35S promoter in Arabidopsis leafprotoplasts. MYB46 activates the C3H14 promoter via a MYB46-responsivecis-element. To illustrate and evaluate such expression from the C3H14promoter, the C3H14 promoter was linked to a coding region forβ-glucuronidase. The C3H14-β-glucuronidase (C3H14-GUS) construct wasused as a reporter gene (positive control) for MYB46 induction ofexpression. Upon dexamethasone (DEX) treatment, the MYB46-GR chimericprotein becomes functional to activate GUS reporter activity driven bythe AtC3H14 promoter. Dexamethasone (DEX) and/or cycloheximide (CHX)were added to the protoplasts to investigate whether the MYB46 candirectly regulate the expression of the CESA genes without new proteinsynthesis. FIG. 2B is a bar graph illustrating relative GUS activitylevels in control, DEX, CHX, and DEX+CHX treated plant cells. FIG. 2Cillustrates transcription of GUS from control, DEX, and DEX−CHX treatedplant cells. Such a analysis shows that DEX-treated MYB46 inducedexpression from the promoters of C3H14 (FIG. 2C) but GUS activity wasinhibited in the presence of 2 μM CHX (FIG. 2B). The expression level ofthe GUS reporter gene in the protoplasts transfected with no effectorconstruct was used as the Control and the GUS expression from thisconstruct was deemed to be 1. Error bars indicate the standard deviationof three biological replicates. FIG. 2D illustrates results of areal-time PCR analysis showing that the DEX activated MYB46-GR fusionprotein directly regulates the expression of CESA4, CESA7 and CESA8genes in the absence of new protein synthesis. Error bars represent thestandard deviation of three biological replicates.

FIGS. 3A and 3B illustrate that MYB46 binds to e promoters of CESA4,CESA7 and CESA8. A GST-MYB46 fusion protein was first incubated with thewild type, double-stranded ³²P-labeled oligodeoxynucleotides with thewild type CESA4, CESA7 and CESA8 promoter sequences shown in FIG. 3A.The GST protein was used as control protein. Unlabeled competitorpromoter oligonucleotides were then added the assays to generate theassay mixtures identified FIG. 3B, where the competitors wereoligonucleotides with wild type and mutated CESA4, CESA7 and CESA8promoter sequences shown in FIG. 3A. FIG. 3A shows the CESA4 wild typepromoter sequence that included SEQ ID NO:3 (ProCesA4wt) the CESA4mutated promoter sequences that included SEQ ID NO:4 (ProCesA4m1) andSEQ NO:5 (ProCesA4m2); the CESA7 wild type promoter sequence thatincluded SEQ ID NO:6 (ProCesA7wt); the CESA7 mutated promoter sequencesthat included SEQ ID NO:7 (ProCesA7m1) and SEQ ID NO: 8 (ProCesA7m2);the CESA8 wild type promoter sequence that included SEQ ID NO:9(ProCesA8wt); and the CESA8 mutated promoter sequences that included SEQID NO:10 (ProCesA8m1) and SEQ ID NO:11 (ProCesA8m2) (dashes indicate nosequence difference). To generate the results shown FIG. 3B, each assaymixture was then subjected to an electrophoretic mobility shift assay(EMSA) by polyacrylamide gel electrophoresis (PAGE). Complexes formedbetween labeled wild type promoters and the MYB46 protein migrated moreslowly than the non-complexed promoter oligonucleotides, and thecomplexes were detectable if the unlabeled wild type or mutant promoterdid not displace the labeled promoter oligonucleotide. FIG. 3B showsthat the GST-MYB46 fusion protein binds to CESA4, CESA7 and CESA8promoter fragments, resulting in retardation of the mobility. Thepromoter regions used for the DNA probes in each experiment areindicated below the gel images. Competition for the protein-DNA bindingwas performed using 60× unlabeled probes. The free unbound DNA probesare indicated by the arrow.

FIG. 4A-B illustrate a chromatin immunoprecipitation (ChIP) analysis ofMYB46 binding to the CESA promoter sequences in vivo. FIG. 4A is adiagram of the construct (vector) used for the inducible expression ofMY846-GFP. FIG. 4B illustrates the results of a real-time quantitativePCR analysis showing the enrichment of the C3H14 and CESA4, CESA7 andCESA8 promoter sequences after chromatin immunoprecipitation. The valueswere normalized against that of the control DNA (MYB46 promoter). C3H14and MYB54 promoters were used as positive and negative control,respectively. Error bars represent the standard deviation of threebiological replicates.

FIG. 5A-B illustrate changes in cell wall crystalline cellulosecomposition detected when MYB46 is over-expressed in wild typeArabidopsis (WT) and transgenic Arabidopsis plants. The transgenicArabidopsis plants OX8 and OX9 over-express the MYB46 transcriptionfactor, while the DEX transgenic Arabidopsis plant has adexamethasone-inducible MYB46 transgene. MYB46 expression in the DEXplants was induced by 24 hr of dexamethasone treatment (+DEX). As acontrol, a DEX plant was mock-treated for 24 hr with 0.05% ethanol andof 0.02% silwet surfactant (−DEX). FIG. 5A is a bar graph showing thecell wall crystalline cellulose content from 3-weeks old Arabidopsisleaves of the indicated plant types. Crystalline cellulose content wasincreased in the OX8 and OX9 leaves that over-express MYB46, as well asin the MYB46 dexamethasone (−DEX) inducible leaves. FIG. 5B shows imagesof eight-week old Arabidopsis stem sections, where crystalline cellulosewas detected by a carbohydrate-binding module (CBM3a) by indirectimmunofluorescence. Scale bars=50 μm. The images are labeled with theplant types, with the exception that the image identified as −CBM3a hadno CBM3a label. The arrows illustrate that MYB46 over-expression in theOX8 and OX9 plants gives rise to intensive cellulose recognition byCBM3a in the walls of epidermal cells compared with those of wild type(WT).

FIG. 6 illustrates to which CESA promoters the HAM1 and HAM2 factorsbind, as detected by electrophoretic mobility shift assays. As shown,both HAM1 and HAM2 bind to the CESA4 promoter in the region ofnucleotide position −666 to −294 upstream from the coding region ofCESA4. The HAM2 factor also bound to the CESA7 promoter in the region ofnucleotide position −260 to −1 upstream from the coding region of CESA7.Procedures similar to those described above for FIG. 3 were employed.

FIG. 7 illustrates to which CESA promoters the MYB112 factor binds, asdetected by electrophoretic mobility shift assays. As shown, the MYB112factor binds to the CESA4 promoter in the region of nucleotide position−666 to −294 upstream from the coding region of CESA4. Proceduressimilar to those described above for FIG. 3 were employed.

FIG. 8 illustrates to which CESA promoters the WRKY11 factor binds, asdetected by electrophoretic mobility shift assays. As shown, the WRKY11factor binds to the CESA4 promoter in the region of nucleotide position−666 to −294 upstream from the coding region of CESA4. Proceduressimilar to those described above for FIG. 3 were employed.

FIG. 9 illustrates to which CESA promoters the ERF6 factor binds, asdetected by electrophoretic mobility shift assays. As shown, the ERF6factor binds to the CESA4 promoter in the region of nucleotide position−666 to −294 upstream from the coding region of CESA4 Procedures similarto those described above for FIG. 3 were employed.

FIG. 10 is a schematic diagram showing T-DNA insertion sites in thecesa4, cesa7 and cesa8 mutants. The gray boxes represent exons, theblack bars between the gray boxes represent introns and the white boxesrepresent UTRs. The black arrowheads indicate the site of T-DNAinsertion.

FIG. 11 is a schematic diagram showing point mutations n the promotersof CESA4, CESA7 and CESA8 (SEQ ID NO: 65-78). The vertical arrowsindicate the locations of the mutation points and the sequences shownare listed in Table 2.

FIG. 12 shows electrophoretically separated amplicons confirming geneticcomplementation in the transgenic Arabidopsis plants by genomic DNA PCR.WT, wild-type; VC, vector control; M, cesa T-DNA insertion mutants; WC,genetic complementation of the mutants with native promoter-driven CESACDS; MC, genetic complementation of the mutants with mutatedpromoter-driven CESA CDS; T-DNA, the amplified DNA fragment with theT-DNA left border primer and the CESA4, CESA7 and CESA8 primers flankingthe T-DNA insertion site; Endogenous, the amplified CESA4, CESA7 andCESA8 DNA fragments by the forward and reverse CESA primers (Table 1).

FIG. 13A-D illustrates that MYB46 is required for functional expressionof CESA4, CESA7, and CESA8 in Arabidopsis. FIG. 13A is a schematicdiagram of the constructs for expression of CESA coding regions drivenby either a native (WT) or mutated promoter. The mRNA expression levelsfrom these promoters operably linked to cesa4, cesa7 and cesa8 codingregions are shown in FIG. 13B-D. FIG. 13B shows images of wild type,control and transgenic Arabidopsis plants expressing CESA4 from themutant (M) and native (WC) promoters. FIG. 13C shows images of wildtype, control and transgenic Arabidopsis plants expressing CESA7 fromthe mutant (M) and native (WC) promoters. FIG. 13D shows images of wildtype, control and transgenic Arabidopsis plants expressing CESA8 fromthe mutant (M) and native (WC) promoters. WT, wild-type (Col-0); VC,vector control (pCB308); M, T-DNA insertion mutants (cesa4, cesa7 andcesa8); WC, genetic complementation of the mutants with nativepromoter-driven CESA CDS; MC, genetic complementation of the mutantswith mutated promoter-driven CESA CDS. Images are a representative of atleast 15 plants observed in each wild-type and transgenic lines. Thepanels below the images of plants show electrophoretically separatedRT-PCR products illustrating the expression of the CESAs. Total RNAs(500 ng) was extracted from 5-week-old stems and quantitativelyamplified by RT-PCR (28-31 cycles of amplification). Actin was used as acontrol.

FIG. 14 shows stem cross-sections of wild-type, vector control, cesaT-DNA insertion mutants and their genetic complementations. All of thestems from the three mutants (cesa4, cesa7 and cesa8) show collapsedxylem phenotype. Stems from genetic complementation with nativepromoter-driven CESA CDSs recovered normal xylem phenotype, while thosewith mutated promoter-driven CDSs failed to do so. Arrows indicatecollapsed xylem cells. Ph, phloem; Xy, xylem. Size bars represent 50 μm.Images are a representative of at least 15 plants observed in eachwild-type and transgenic lines.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to nucleic acids, proteins and methods useful formodulating the quality and quantity of cellulose in plants. Plants withsuch altered cellulose structure/content are useful sources of fiber,lumber and paper. In addition, plants with such altered cellulosestructure/content may be hardier and less prone to damage byenvironmental forces (e.g., wind).

Cellulose

Cellulose is a major component of plant fiber and is composed ofcrystalline beta-1,4-glucan microfibrils. It is a polysaccharide withthe formula (C₆H₁₀O₅)_(n), where n is an integer of from 100-200,000.Thus, in general, cellulose consists of a linear chain of severalhundred to over ten thousand β(1→4) linked D-glucose units. The β(1→4)linkage is distinct from the α(1→4)-glycosidic bonds present in starch,glycogen, and other carbohydrates. Unlike starch, cellulose is astraight chain polymer without coiling and branching. Instead, cellulosehas an extended and substantially stiff rod-like conformation, wherehydroxyl groups on the glucose from one chain form hydrogen bonds withoxygen molecules on the same or on a neighbor chains, holding the chainsfirmly together side-by-side and forming microfibrils.

These microfibrils are strong and can resist enzymatic and mechanicaldegradation. For example, by virtue of its ability to formsemicrystalline microfibrils, the tensile strength of celluloseapproaches that of some metals. Niklas, PLANT BIOMECHANICS: ANENGINEERING APPROACH TO PLANT FORM AND FUNCTION, The University ofChicago Press, p. 607 (1992). However, the bending strength of the culmof normal and brittle-culm mutants of barley has been found to bedirectly correlated with the concentration of cellulose in the cellwall. Kokubo, et al., (1989), Plant Physiology 91:876-882; Kokubo, etal., (1991) Plant Physiology 97:509-514.

Cellulose Synthases

Cellulose is synthesized by multimeric cellulose synthase (CESA)complexes at the plasma membrane (Somerville, 2006). In plants, twodistinct groups of CESAs (each consisting of at least three differentisoforms) are preferentially and coordinately expressed during primaryand secondary cell wall deposition (Endler and Persson, 2011). TheArabidopsis genome contains 10 CESA genes (Pear et al., 1996; Richmondand Somerville, 2001). Recently, a cellulose synthase-interactiveprotein (CSI1) has been identified as a non-CESA component of the CESAcomplexes (Gu et al., 2010). Several proteins, such as KORRIGAN, COBRA,KOBITO1, are also known to negatively affect the synthesis of cellulosewhen mutated or misregulated (Endler and Persson, 2011).

Analyses of various cellulose synthesis mutants has revealed that CESA1,CESA3, CESA6, CESA2, CESA5, and CESA9 (Arioli et al., 1998; Fagard etal., 2000; Scheible et al., 2001; Desprez et al., 2002 and 2007; Perssonet al., 2007) are associated with the CESA complexes that are activeduring primary wall formation.

In contrast, CESA4, CESA7, and CESA8 are necessary for secondary wallcellulose biosynthesis (Turner and Somerville, 1997; Taylor et al.,1999; 2000; 2003; Doblin et al., 2002; Williamson et al., 2002). Unlikethe primary wall CESA complex, the three secondary wall CESA subunitsappear to be equally important in the function of the complex in xylemvessels and cannot substitute for each other (Gardiner et al., 2003).None of the proteins has been reported to be directly associated withthe CESA complex, suggesting that their effects on cellulose synthesismay be indirect. However, as shown herein, the expression of CESA4,CESA7, and CESA8 is directly regulated by binding of the transcriptionfactor MYB46 to cis-acting regulatory motifs that reside in the promoterregions of the CESA4, CESA7 and CESA8 genes. In fact, MYB46 is a keyfactor that can increase CESA4, CESA7 and CESA8 gene expression.

Control of Cellulose Synthase Expression

Formation of secondary wall requires a coordinated transcriptionalactivation of the genes involved in the biosynthesis of secondary wallcomponents such as cellulose, hemicellulose and lignin. Recent studieson transcription factors have provided some insight into the complexprocess of transcriptional regulation of secondary wall biosynthesis(Demura and Ye, 2010; Ko et al. 2007 and 2009; Mitsuda et al, 2005;Mitsuda et al., 2007; Zhong and Ye, 2007; Zhong et al., 2007, 2008, and2010).

However, while CESAs appear to be the only group of proteins with theability to synthesize new cellulose molecules, until the presentinvention little was known about how secondary wall-associated CESAgenes were regulated. Prior to the invention described herein, notranscription factor binding, to any CESA promoter had yet beenreported.

The data described herein shows that several transcription factorsselectively bind to discrete CESA promoters and that CESA production maybe a rate limiting factor for cellulose biosynthesis. The transcriptionfactors active in production of CESAs include MYB46, HAM1, HAM2, MYB112,WRKY11, ERF6, as well as other transcription factors with at least 70%,or at least 75%, or at least 80%, or at least 85%, or at least 90%, orat least 95%, or at least 97% sequence identity to any of SEQ ID NO:1,12, 14, 16, 18, and 20.

MYB46 Transcription Factor

For example, as illustrated herein, over-expression of the MYB46transcription factor results in ectopic deposition of secondary walls inthe cells that are normally parenchymatous, while suppression of MYB46function reduces secondary wall thickening. Knockout of MYB46 functioneffectively knocks out CESA4, CESA7 and CESA8 gene expression.

The MYB46 transcription factor sequence is available from the NationalCenter for Biotechnology Information (NCBI) database (see, e.g., thewebsite at ncbi.nlm.nih.gov). For example, a nucleic acid sequence forthe MYB46 transcription factor is available as accession numberAT5G12870, and reproduced below as SEQ ID NO:1.

1 ATGAGGAAGC CAGAGGTAGC CATTGCAGCT AGTACTCACC 41AAGTAAAGAA GATGAAGAAG GGACTTTGGT CTCCTGAGGA 81AGACTCAAAG CTGATGCAAT ACATGTTAAG CAATGGACAA 121GGATGTTGGA GTGATGTTGC GAAAAACGCA GGACTTCAAA 161GATGTGGCAA AAGCTGCCGT CTTCGTTGGA TCAACTATCT 201TCGTCCTGAC CTCAAGCGTG GCGCTTTCTC TCCTCAAGAA 241GAGGATCTCA TCATTCGCTT TCATTCCATC CTCGGCAACA 281GGTGGTCTCA GATTGCAGCA CGATTGCCTG GTCGGACCGA 321TAACGAGATC AAGAATTTCT GGAACTCAAC AATAAAGAAA 361AGGCTAAAGA AGATGTCCGA TACCTCCAAC TTAATCAACA 401ACTCATCCTC ATCACCCAAC ACAGCAAGCG ATTCCTCTTC 441TAATTCCGCA TCTTCTTTGG ATATTAAAGA CATTATAGGA 481AGCTTCATGT CCTTACAAGA ACAAGGCTTC GTCAACCCTT 541CCTTGACCCA CATACAAACC AACAATCCAT TTCCAACGGG 581AAACATGATC AGCCACCCGT GCAATGACGA TTTTACCCCT 601TATGTAGATG GTATCTATGG AGTAAACGCA GGGGTACAAG 641GGGAACTCTA CTTCCCACCT TTGGAATGTG AAGAAGGTGA 681TTGGTACAAT GCAAATATAA ACAACCACTT AGACGAGTTG 721AACACTAATG GATCCGGAAA CGCACCTGAG GGTATGAGAC 761CAGTGGAAGA ATTTTGGGAC CTTGACCAGT TGATGAACAC 801TGAGGTTCCT TCGTTTTACT TCAACTTCAA ACAAAGCATA 841 TGAThe amino acid sequence of the MYB46 polypeptide encoded by the SEQ IDNO:1 nucleic acid is as follows (SEQ ID NO:2).

1 MRKPEVAIAA STHQVKKMKK GLWSPEEDSK LMQYMLSNGQ 41GCWSDVAKNA GLQRCGKSCR LRWINYLRPD LHRGAFSPQE 121EDLIIRFHSI LGNRWSQIAA RLPGRTDNEI KNFWNSTIKK 161RLKKMSDTSN LINNSSSSPN TASDSSSNSA SSLDIKDIIG 201SFMSLQEQGF VNPSLTHIQT NNPFPTGNMI SHPCNDDFTP 241YVDGIYGVNA GVQGELYFPP LECEEGDWYN ANINNHLDEL 281NTNGSGNAPE GMRPVEEFWD LDQLMNTEVP SFYFNFKQSI

Experimental evidence is provided herein showing that MYB46 can directlyregulate the expression of three secondary wall cellulose synthases(CESA4, CESA7 and CESA8) in Arabidopsis plants. Genome-wide analysis ofpromoter sequences in Arabidopsis by the inventors has revealed thatmany secondary wall biosynthetic genes, including CESA4, CESA7 andCESA8, have one or more cis-acting regulatory motifs (named ‘M46REs’) intheir promoter regions. As demonstrated herein, MYB46 binds to suchcis-acting regulatory motifs and stimulates expression of the secondarywall biosynthetic genes CESA4, CESA7 and CESA8.

One cis-acting regulatory motif that is recognized by MYB46 is naturallylocated in the promoter region of CESA4, and has the following sequence.

TCACTCACAG TTTGGTACAA CCTCA (SEQ ID NO: 3 also called ProCesA4wt).Two mutant cis-acting CESA4 regulatory motifs with point mutations havebeen tested and do not bind MYB46. These mutant cis-acting CESA4regulatory motifs have the following sequences.

TCACTCACAG T G TGGTACAA CCTCA (SEQ ID NO: 4; also called ProCesA4m1).TCACTCACAG TTT T GTACAA CCTCA (SEQ ID NO: 5; also called ProCesA4m2).

Another cis-acting regulatory motif that is recognized by MYB46 isnaturally located in the promoter region of CESA7, and has the followingsequence.

CAGAAAATTCACCTAATTAACGACA (SEQ ID NO: 6; also called ProCesA7wt).Two mutant cis-acting CESA7 regulatory motifs with point imitations havebeen tested and do not bind MYB46. These mutant cis-acting CESA7regulatory motifs have the following sequences.

CAGAAAATTCACCT G ATTAAGGACA (SEQ ID NO: 7; also called ProCesA7m1).CAGAAAATTCAC A TAATTAAGGACA (SEQ ID NO: 8; also called ProCesA7m2).

Another cis-acting regulatory motif that is recognized by MYB46 isnaturally located in the promoter region of CESA8, and has the followingsequence.

CTTATAGAAAGTTGGTGATTGAAAA (SEQ ID NO: 9; also called ProCesA8wt).Two mutant cis-acting CESA8 regulatory motifs with point mutations havebeen tested and do not bind MYB46. These mutant cis-acting CESA8regulatory motifs have the following sequences.

CTTATAGAAAG G TGGTGATTGAAAA (SEQ ID NO: 10; also called ProCesA8m1).CTTATAGAAAGTT T GTGATTGAAAA (SEQ ID NO: 11; also called ProCesA8m2).Nucleic acids encoding these wild type and mutant cis-acting regulatorymotifs are therefore useful targets for regulated gene expression.HAM1 and HAM2 Transcription Factors

As illustrated herein, the HAM1 and HAM2 transcription factorsselectively bind to some, but not all cellulose synthase promoters. Forexample, the HAM1 binds to the regions of the CESA4 promoter, while theHAM2 transcription factor binds to regions of both the CESA4 promoterand the CESA7 promoter.

A nucleotide sequence for the HAM1 transcription factor is shown below(SEQ ID NO:12).

1 ATGGGATCGT CTGCGGATAC AGAGACGGCG ATGATAATCG 41CCACACCGGC GTCGAACCAT AATAATCCGG CAACCAACGG 81CGGAGATGCG AATCAGAATC ATACTTCTGG TGCGATACTC 121GCTCTCACGA ATTCAGAATC GGATGCTTCG AAGAAGAGAA 161GAATGGGGGT GCTTCCGCTC GAGGTTGGTA CTCGCGTGAT 201GTGTCAATGG AGAGACGGAA AATACCATCC GGTGAAGGTT 241ATCGAGCGCC GAAAGAATTA TAATGGTGGT CACAATGATT 231ACGAGTACTA CGTTCATTAC ACAGAGTTTA ATAGAAGATT 321GGATGAATGG ATTAAGCTTG AACAGCTTGA CCTTGATTCA 361GTAGAGTGTG CTTTAGATGA AAAAGTTGAA GACAAGGTGA 401CTAGCTTGAA GATGACACGA CACCAGAAAC GGAAGATTGA 441TGAGACTCAT GTAGAGGGTC ATGAAGAGCT GGATGCTGCC 481AGTTTGCGTG AACACGAGGA GTTCACGAAA GTGAAGAACA 521TAGCTACGAT TGAGCTTGGG AAGTATGAGA TTGAGACGTG 561GTACTTCTCT CCTTTTCCTC CAGAATACAA TGACTGCGTG 601AAGCTCTTTT TCTGTGAGTT TTGCCTCAGT TTTATGAAGC 641GCAAAGAGCA GCTTCAAAGA CATATGAGGA AATGCGATTT 681GAAGCACCCC CCTGGGGATG AAATCTATCG AAGCTCTACT 721TTGTCAATGT TTGAGGTGGA TGGCAAGAAG AATAAGGTCT 761ATGCACAGAA CCTCTGTTAT CTGGCAAAGT TATTTCTTGA 801CCACAAAACT CTTTACTATG ACGTTGATTT GTTCCTGTTC 841TATATTCTCT GTGAATGTGA TGATCGTGGA TGCCACATGG 881TTGGATACTT TTCAAAGGAA AAACACTCAG AAGAAGCTTA 921CAACTTGGCT TGCATCCTTA CACTTCCTCC ATATCAAAGG 961AAGGGCTATG GCAAATTCTT AATAGCCTTC TCCTATGAAC 1001TCTCAAAGAA AGAGGGCAAA GTCGGGACAC CGGAAAGGCC 1041GCTCTCTGAT CTAGGGTTAG TGAGTTACAG AGGTTACTGG 1081ACTCGGATTT TATTAGACAT TTTGAAAAAG CACAAGGGAA 1121ACATATCTAT CAAGGAGCTG AGCGACATGA CAGCGATTAA 1161AGCAGAAGAT ATATTAAGCA CCCTGCAGAG CTTGGAACTG 1201ATACAATACA GGAAAGGACA ACACGTAATC TGCGCGGATC 1241CTAAGGTACT GGACCGACAC TTGAAAGCGG CAGGCCGAGG 1231TGGTCTTGAT GTGGATGTGA GCAAAATGAT ATGGACTCCT 1321 TACAAAGAGC AGAGCTAA

An amino acid sequence for the HAM1 transcription factor encoded by theSEQ ID NO:12 nucleic acid is shown below as SEQ ID NO:13.

1 MGSSADTETA MIIATPASNH NNPATNGGDA NQNHTSGAIL 41ALTNSESDAS KKRRMGVLPL EVGTRVMCQW RDGKYHPVKV 81IERRKNYNGG HNDYEYYVHY TEFNRRLDEW IKLEQLDLDS 121VECALDEKVE DKVTSLKMTR HQKRKIDETH VEGHEELDAA 161SLREHEEFTK VKNIATIELG KYEIETWYFS PFPPEYNDCV 201KLFFCEFCLS FMKRKEQLQR HMRKCDLKHP PGDEIYRSST 241LSMFEVDGKK NKVYAQNLCY LAKLFLDHKT LYYDVDLFLF 281YILCECDDRG CHMVGYFSKE KHSEEAYNLA CILTLPPYQR 321KGYGKFLIAF SYELSKKEGK VGTPERPLSD LGLVSYRGYW 361TRILLDILKK HKGNISIKEL SDMTAIKAED ILSTLQSLEL 401IQYRKGQHVI CADPKVLDRH LKAAGRGGLD VDVSKMIWTP 441 YKEQSExperiments described herein demonstrate that the HAM1 transcriptionfactor binds to the CESA4 promoter in the region of nucleotide position−294 to −666 upstream of the coding region of the CESA4 gene.

A nucleotide sequence for the HAM2 transcription factor is shown below(SEQ ID NO:14).

1 ATGGGATCGT CAGCGAATAC AGAAACCAAC GGCAACGCAC 41CGCCACCGTC GTCGAATCAA AAGCCTCCGG CTACGAACGG 81CGTTGATGGG TCTCATCCTC CTCCTCCTCC TTTAACTCCT 121GATCAAGCTA TTATAGAGTC GGATCCGTCG AAGAAGAGGA 161AAATGGGGAT GCTTCCTCTA GAAGTGGGTA CTCGTGTGAT 201GTGTCGGTGG AGAGACGGGA AACACCATCC GGTGAAAGTA 241ATTGAGCGCC GGCGGATACA TAACGGCGGT CAAAATGATT 281ACGAGTATTA CGTTCATTAC ACTGAGTTTA ATAGGAGGCT 321GGATGAATGG ACTCAGCTGG ACCAACTGGA CCTTGATTCA 361GTAGAGTGCG CTGTAGATGA AAAAGTGGAA GACAAGGTAA 401CAAGCTTGAA GATGACACGT CACCAGAAGA GGAAGATCGA 441TGAGACACAT ATAGAGGGTC ATGAAGAGCT GGATGCAGCA 481AGTTTGCGTG AACATGAAGA GTTCACGAAA GIGAAGAACA 521TATCAACAAT TGAGCTTGGA AAATATGAGA TTGAGACTTG 561GTACTTCTCC CCTTTTCCGC CAGAATACAA TGAGTGTGTG 601AAGCTCTTTT TTTGTGAGTT TTGCCTGAAC TTCATGAAAC 641GCAAAGAGCA GCTTCAAAGG CATATGAGGA AGTGTGACCT 681GAAGCACCCA CCTGGTGATG AAATTTACCG AAGTGGTACC 721TTGTCAATGT TTGAGGTAGA TGGCAAAAAG AACAAGGTTT 761ATGCACAGAA TCTCTGCTAC CTGGCAAAGT TATTTCTTGA 801CCACAAAACT CTTTACTACG ATGTTGATTT GTTTCTATTC 841TACGTTCTTT GCGAATGTGA TGACCGAGGA TGCCACATGG 881TTGGGTACTT TTCAAAGGAG AAGCATTCGG AAGAAGCATA 921CAACTTAGCT TGCATTCTAA CCCTGCCTTC AIATCAAAGA 961AAAGGCTATG GAAAGTTCTT AATAGCCTTT TCCTATGAAC 1001TGTCAAAGAA AGAGGGAAAA GTTGGGACAC CGGAAAGACC 1041CTTGTCGGAT CTAGGCTTAC TAAGCTACAG AGGTTATTGG 1081ACTCGTGTTC TATTAGAAAT CTTGAAAAAA CATAAGGGAA 1121ACATTTCTAT CAAGGAGCTG AGCGACGTGA CAGCAATCAA 1161AGCGGAAGAT ATATTAAGCA CACTTCAGAG CCTAGAACTG 1201ATACAGTACA GGAAAGGACA GCATGTGATC TGTGCGGATC 1241CAAAGGTTCT GGACCGACAT CTGAAAGCTG CAGGCCGAGG 1281TGGTCTTGAT GTAGATGCTA GCAAACTGAT TTGGACACCT 1321 TACAAGGAGC AGAGTTAA

An amino acid sequence for the HAM2 transcription factor encoded by theSEQ ID NO:14 nucleic acid is shown below as SEQ ID NO:15.

1 MGSSANTETN GNAPPPSSNQ KPPATNGVDG SHPPPPPLTP 41DQAIIESDPS KKRKMGMLPL EVGTRVMCRW RDGKHHPVKV 81IERRRIHNGG QNDYEYYVHY TEFNRRLDEW TQLDQLDLDS 121VECAVDEKVE DKVTSLKMTR HQRKIDETH IEGHEELDAA 161SLREHEEFTK VKNISTIELG KYEIETWYFS PFPPEYNDCV 201KLFFCEFCLN FMKRKEQLQR HMRKCDLKHP PGDEIYRSGT 241LSMFEVDGKK NKVYAQNLCY LAKLFLDHKT LYYDVDLFLF 281YVLCECDDRG CHMVGYFSKF KHSEEAYNLA CILTLPSYQR 321KGYGKFLIAF SYELSKKEGK VGTPERPLSD LGLLSYRGYW 361TRVLLEILKK HKGNISIKEL SDVTAIKAED ILSTLQSLEL 401IQYRKGQHVI CADPKVLDRH LKAAGRGGLD VDASKLIWTP 441 YKDQS

Experiments described herein demonstrate that, like the HAM1transcription factor, the HA M2 transcription factor binds to the CESA4promoter in the region of nucleotide position −294 to −666 upstream ofthe coding region of the CESA4 gene. In addition, the HAM2 transcriptionfactor binds to the CESA7 promoter in the region of nucleotide position−1 to −260 upstream of the coding region of the CESA7 gene.

MYB112 Transcription Factor

As illustrated herein, the MYB112 transcription factor selectively bindsto some, but not all cellulose synthase promoters. For example, theMYB112 binds selectively only to regions of the CESA4 promoter.

A nucleotide sequence for the MYB112 transcription factor is shown below(SEQ ID NO:16).

1 ATGAATATAA GTAGAACAGA ATTCGCAAAC TGTAAAACCC 41TTATAAATCA TAAAGAAGAA GTCGAAGAAG TCGAGAAAAA 81GATGGAAATA GAAATAAGGA GAGGTCCATG GACTGTGGAA 121GAAGACATGA AGCTCGTCAG TTACATTTCT CTTCACGGTG 161AAGGAAGATG GAACTCCCTC TCTCGTTCTG CTGGACTGAA 201TAGAACGGGG AAAAGTTGCA GATTGCGGTG GCTAAATTAT 241CTCCGGCCGG ATATCCGCCG TGGAGACATA TCCCTTCAAG 281AACAATTTAT CATCCTTGAA CTCCATTCTC GTTGGGGAAA 321TCGGTGGTCA AAGATTGCTC AACATTTACC GGGAAGAACA 361GATAACGAGA TAAAGAATTA TTGGAGAACA CGTGTTCAAA 401AGCATGCAAA ACTTCTAAAA TGTGACGTGA ACAGCAAGCA 441ATTCAAAGAC ACCATCAAAC ATCTCTGGAT GCCTCGTCTC 481ATCGAGAGAA TCGCCGCCAC TCAAAGTGTC CAATTTACCT 521CTAACCACTA CTCGCCTGAG AACTCCAGCG TCGCCACCGC 561CACGTCATCA ACGTCGTCGT CTGAGGCTGT GAGATCGAGT 601TTCTACGGTG GTGATCAGGT GGAATTTGGA ACGTTGGATC 641ATATGACAAA TGGTGGTTAT TGGTTCAACG GCGGAGATAC 681GTTTGAAACT TTGTGTAGTT TTGACGAGCT CAACAAGTGG 721 CTCATACAGT AG

An amino acid sequence for the MYB112 transcription factor encoded bythe SEQ ID NO:16 nucleic acid is shown below as SEQ ID NO:17.

1 MNISRTEFAN CKTLINHKEE VEEVEKKMEI EIRRGPWTVE 41EDMKLVSYIS LHGEGRWNSL SRSAGLNRTG KSCRLRWLNY 81LRPDIRRGDI SLQEQFIILE LHSRWGNRWS KIAQHLPGRT 121DNEIKNYWRT RVQKHAKLLK CDVNSKQFKD TIKHLWMPRL 161IERIAATQSV QFTSNHYSPE NSSVATATSS TSSSEAVRSS 201FYGGDQVEFG TLDHMTNGGY WFNGGDTFET LCSFDELNKW 241 LIQExperiments described herein demonstrate that the MYB112 transcriptionfactor binds to the CESA4 promoter in the region of nucleotide position−294 to −666 upstream of the coding region of the CESA44 gene.WRKY11 Transcription Factor

As illustrated herein, the WRKY11 transcription factor selectively bindsto some, but not all cellulose synthase promoters. For example, theWRKY11 binds selectively only to regions of the CESA4 promoter.

A nucleotide sequence for the WRKY11 transcription factor is shown below(SEQ ID NO:18).

1 ATGGCCGTCG ATCTAATGCG TTTCCCTAAG ATAGATGATC 41AAACGGCTAT TCAGGAAGCT GCATCGCAAG GTTTACAAAG 81TATGGAACAT CTGATCCGTG TCCTCTCTAA CCGTCCCGAA 121CAACAACACA ACGTTGACTG CTCCGAGATC ACTGACTTCA 161CCGTTTCTAA ATTCAAAACC GTCATTTCTC TCCTTAACCG 201TACTGGTCAC GCTCGGTTCA GACGCGGACC GGTTCACTCC 241ACTTCCTCTG CCGCATCTCA GAAACTACAG AGTCAGATCG 281TTAAAAATAC TCAACCTGAG GCTCCGATAG TGAGAACAAC 321TACGAATCAC CCTCAAATCG TTCCTCCACC GTCTAGTGTA 361ACACTCGATT TCTCTAAACC AAGCATCTTC GGCACCAAAG 401CTAAGAGCGC CGAGCTGGAA TTCTCCAAAG AAAACTTCAG 441TGTTTCTTTA AACTCCTCAT TCATGTCGTC GGCGATAACC 481GGAGACGGCA GCGTCTCCAA TGGAAAAATC TTCCTTGCTT 521CTGCTCCGTT GCAGCCTGTT AACTCTTCCG GAAAACCACC 561GTTGGCTGGT CATCCTTACA GAAAGAGATG TCTCGAGCAT 601GAGCACTCAG AGAGTTTCTC CGGAAAAGTC TCCGGCTCCG 641CCTACGGAAA GTGCCATTGC AAGAAAAGCA GGAAAAATCG 681GATGAAGAGA ACCGTGAGAG TACCGGCGAT AAGTGCAAAG 721ATCGCCGATA TTCCACCGGA CGAATATTCG TGGAGGAAGT 761ACGGACAAAA ACCGATCAAC GGCTCACCAC ACCCACGTGG 801TTACTACAAG TGCAGTACAT TCAGAGGATG TCCAGCGAGG 841AAACACGTGG AACGAGCATT AGATGATCCA GCGATGCTTA 881TTGTGACATA CGAAGGAGAG CACCGTCATA ACCAATCCGC 921GATGCAGGAG AATATTTCTT CTTCAGGCAT TAATGATTTA 961 GTGTTTGCCT CGGCTTGAAn amino acid sequence for the WRKY11 transcription factor encoded bythe SEQ ID NO:18 nucleic acid is shown below as SEQ ID NO:19.

1 MAVDLMRFPK IDDQTAIQEA ASQGLQSMEH LIRVLSNRPE 41QQHNVDCSEI TDFTVSKFKT VISLLNRTGH ARFRRGRVHS 81TSSAASQKLQ SQIVKNTQPE APIVRTTTNH PQIVPPPSSV 121TLDFSKPSIF GTKAKSAELE FSKENFSVSL NSSFMSSAIT 161GDGSVSNGKI FLASAPLQPV NSSGKPPLAG HPYRKRCLEH 201EHSESFSGKV SGSAYGKCHC KKSRKNRMKR TVRVPAISAK 241IADIPPDEYS WRKYGQKPIK GSPHPRGYYK CSTFRGCPAR 281KHVERALDDP AMLIVTYEGE HRHNQSAMQE NISSSGINDL 321 VFASA

Experiments described herein demonstrate that the WRKY11 transcriptionfactor binds to the CESA4 promoter in the region of nucleotide position−294 to −666 upstream of the coding region of the CESA4 gene.

ERF6 Transcription Factor

As illustrated herein, the ERF6 transcription factor selectively bindsto some, but not all cellulose synthase promoters. For example, the ERF6binds selectively only to regions of the CESA4 promoter.

A nucleotide sequence for the ERF6 transcription factor is shown below(SEQ ID NO:20).

1 ATGGCTACAC CAAACGAAGT ATCAGCTCTT TTCCTCATCA 41AGAAGTATCT CCTCGACGAA TTGTCTCCGT TGCCTACTAC 81TGCCACCACC AATCGATGGA TGAACGATTT CACGTCATTT 121GATCAAACCG GTTTCGAGTT TTCTGAATTT GAAACCAAAC 161CGGAAATAAT CGATCTCGTC ACTCCCAAAC CGGAGATTTT 201TGATTTCGAT GTGAAATCTG AAATTCCATC TGAATCGAAC 241GATTCCTTCA CGTTCCAATC GAATCCTCCT CGCGTTACTG 281TTCAATCCAA TCGAAAACCG CCGTTGAAGA TCGCACCACC 321GAACCGAACC AAGTGGATTC AATTCGCAAC CGGAAATCCT 361AAACCGGAAC TTCCCGTACC GGTTGTAGCA GCAGAGGAGA 401AGAGGCATTA CAGAGGAGTG AGGATGAGGC CGTGGGGGAA 441ATTCGCGGCG GAGATTCGAG ACCCGACTCG TCGTGGAACT 481CGTGTTTGGC TCGGGACGTT TGAGACGGCG ATCGAAGCGG 521CTAGAGCTTA CGACAAAGAA GCGTTTAGAC TACGAGGATC 561AAAGGCGATT CTGAATTTCC CGCTTGAAGT TGACAAGTGG 601AATCCACGCG CTGAAGATGG TCGTGGCCTG TACAACAAAC 641GGAAGAGAGA CGGCGAGGAG GAGGAAGTGA CGGTGGTTGA 681GAAAGTGCTA AAGACGGAGG AGAGTTACGA CGTTAGCGGC 721GGCGAGAATG TTGAGTCAGG TTTGACGGCG ATAGATGACT 761GGGATTTGAC GGAGTTTCTG AGCATGCCGC TTTTATCGCC 801GTTATCTCCA CACCCACCGT TTGGTTATCC ACAATTGACC 841 GTTGTTTGA

An amino acid sequence for the ERF6 transcription factor encoded by theSEQ ID NO:20 nucleic acid is shown below as SEQ ID NO:21.

1 MATPNEVSAL FLIKKYLLDE LSPLPTTATT NRWMNDFTSF 41DQTGFEFSEF ETKPEIIDLV TPKPEIFDFD VKSEIPSESN 81DSFTFQSNPP RVTVQSNRKP PLKIAPPNRT KWIQFATGNP 121KPELPVPVVA AEEKRHYRGV RMRPWGKFAA EIRDPTRRGT 161RVWLGTFETA IEAARAYDKE AFRLRGSKAI LNFPLEVDKW 201NPRAEDGRGL YNKRKRDGEE EEVTVVEKVL KTEESYDVSG 241GENVESGLTA IDDWDLTEFL SMPLLSPLSP HPPFGYPQLT 281 VV

Experiments described herein demonstrate that the ERF6 transcriptionfactor binds to the CESA4 promoter in the region of nucleotide position−294 to −666 upstream of the coding region of the CESA4 gene.

Therefore, the MYB46, HAM1, HAM2, MYB112, WRKY11, and ERF6 transcriptionfactors bind to and thereby modulate the expression of various cellulosesynthases. The following table summarizes to which promoters thetranscription factors bind.

Transcription Factor Cellulose Synthase Gene MYB46 CESA4, CESA7, CESA8HAM1 CESA4 HAM2 CESA4, CESA7 MYB112 CESA4 WRKY11 CESA4 ERF6 CESA4

As described herein, modification of the expression of cellulosesynthases can modify cellulose synthase activity, which can altercellulose fiber quantity, either by producing more or less fiber in aparticular plant species or in a specific organ or tissue of aparticular plant. Modification of cellulose synthase activity canincrease the value of the fiber to the end-user and may improve thestructural integrity of the plant cell wall. In addition, becausecellulose is a major cell wall component, inhibition of cellulosesynthesis may be lethal. Inhibitors of cellulose synthase expressionthat target these cis-acting regulatory motifs may therefore serve asherbicides.

Plants Modified to Contain Transcription Factors and/or PromoterSequences

In order to engineer plants with desired quantities of cellulose, one ofskill in the art can introduce transcription factors or nucleic acidsencoding transcription factors into the plants. Such transcriptionfactors can bind to the promoter regions of cellulose synthases (e.g.,CESA4, CESA7 and CESA8) and stimulate their expression. In someembodiments, the transcription factors can bind to any of SEQ ID NO:3, 6and/or 9 and stimulate the expression of coding sequences that areoperably linked to these SEQ ID NOs. In other embodiments, thetranscription factors can bind to any nucleic acid sequence with atleast 95% sequence identity to SEQ ID NO:3, 6 and/or 9 and stimulate theexpression of coding sequences that are operably linked to nucleic acidswith any of these SEQ ID NOs.

In some embodiments, one of skill in the art can inject transcriptionfactors or nucleic acids encoding such transcription factors into youngplants, or into selected regions of plants. Alternatively, one of skillin the art can generate genetically-modified plants that contain nucleicacids encoding transcription factors within their somatic and/or germcells. In addition, those of skill in the art can use any of thepromoters with any the SEQ ID NO:3, 6, and/or 9 promoter sequences withthe transcription factors to drive the expression of other codingregions of interest, for example, by genetically modifying a plant tocontain a promoter nucleic acid upstream of the coding region ofinterest and an expression cassette that can express the transcriptionfactor. Such genetic modification can be accomplished by proceduresavailable in the art. For example, one of skill in the art can preparean expression cassette or expression vector that can express one or moreencoded transcription factors while the promoter is operably linked to acoding region of interest in a separate expression cassette. Plant cellscan be transformed by the expression cassettes or expression vector, andwhole plants (and their seeds) can be generated from the plant cellsthat were successfully transformed with the promoter and/ortranscription factor nucleic acids. Some procedures for making suchgenetically modified plants and their seeds are described below.

Plants modified to contain the isolated transcription factors describedherein (e.g., expressed from a heterologous promoter and/or from atransgene and/or from an expression cassette) can have increasedcellulose content relative to a wild type plant of the same species thatdoes not have such an isolated transcription factor. For example, plantsexpressing one of the transcription factors described herein from anisolated nucleic acid can have at least about 2%, or at least about 4%,or at least about 5%, or at least about 7%, or at least about 10%, or atleast about 12%, or at least about 13%, or at least about 15%, or atleast about 17%, or at least about 20%, or at least about 22%, or atleast about 25%, or at least about 30% increased cellulose contentcompared to a wild type plant of the same species (without the addedisolated transcription factor).

Promoters: The transcription factor nucleic acids of the invention canbe operably linked to a promoter, which provides for expression of mRNAfrom the transcription factor nucleic acids. The promoter is typically apromoter functional in plants and/or seeds, and can be a promoterfunctional during plant growth and development. A transcription factornucleic acid is operably linked to the promoter when it is locateddownstream from the promoter, to thereby form an expression cassette.

Similarly, a nucleic acid segment encoding any of the cellulose synthasepromoters described herein (e.g., any segment that includes SEQ IDNO:3-11, 65-71, or any segment that includes a sequence with at least95% sequence identity to SEQ ID NO:3-11, 65-71) can be operably linkedto a selected coding region of interest, for example, by inserting thepromoter nucleic acid segment upstream of a coding region nucleic acid.

Promoters regulate gene expression. Promoter regions are typically foundin the flanking DNA upstream from the coding sequence in bothprokaryotic and eukaryotic cells. A promoter sequence provides forregulation of transcription of the downstream gene sequence andtypically includes from about 50 to about 2,000 nucleotide base pairs.Promoter sequences can also contain regulatory sequences such asenhancer sequences that can influence the level of gene expression. Someisolated promoter sequences can provide for gene expression ofheterologous DNAs, that is a DNA different from the native or homologousDNA.

Promoter sequences can be strong or weak, or inducible. A strongpromoter provides for a high level of gene expression, whereas a weakpromoter provides for a very low level of gene expression. An induciblepromoter is a promoter that provides for the turning on and off of geneexpression in response to an exogenously added agent, or to anenvironmental or developmental stimulus. For example, a bacterialpromoter such as the P_(tac) promoter can be induced to vary levels ofgene expression depending on the level of isothiopropylgalactoside addedto the transformed cells.

Promoters can also provide for tissue specific or developmentalregulation. In some embodiments, an isolated promoter sequence that is astrong promoter for heterologous DNAs is advantageous because itprovides for a sufficient level of gene expression for easy detectionand selection of transformed cells and provides for a high level of geneexpression when desired.

In some embodiments, heterologous promoters can be operably linked toone or more cellulose synthase coding sequence segment (e.g., CESA4,CESA7 and/or CESA8), where the heterologous promoter is a strongpromoter, weak promoter, inducible promoter, tissue specific promoter,developmentally regulated promoter, or some combination thereof.

The selected promoter-cellulose synthase construct can be placed in anexpression cassette or expression vector.

Expression cassettes for the transcription factor can include, but arenot limited to, a plant promoter with a sequence such as any of the SEQID NO:3-11, 65-71 (or a combination thereof). Expression cassettes forthe transcription factor can also include, but are not limited to, aplant promoter such as the CaMV 35S promoter (Odell et al., Nature. 313:810-812 (1985)), or others such as CaMV 19S (Lawton et al., PlantMolecular Biology. 9: 315-324 (1987)), nos (Ebert et al., Proc. Natl.Acad. Sci. USA. 84: 5745-5749 (1987)), Adhl (Walker et al., Proc. Natl.Acad. Sci. USA. 84: 6624-6628 (1987)), sucrose synthase (Yang et al.,Proc. Natl. Acad. Sci. USA. 87: 4144-4148 (1990)), α-tubulin, ubiquitin,actin (Wang et al., Mol. Cell. Biol. 12: 3399 (1992)), cab (Sullivan etal., Mol. Gen. Genet. 215: 431 (1989)), PEPCase (Hudspeth et at, PlantMolecular Biology. 12: 579-589 (1989)) or those associated with the Rgene complex (Chandler et al., The Plant Cell. 1: 1175-1183 (1989)).Further suitable promoters include the poplar xylem-specific secondarycell wall specific cellulose synthase 8 promoter, cauliflower mosaicvirus promoter, the Z10 promoter from a gene encoding a 10 kD zeinprotein, a Z27 promoter from a gene encoding a 27 kD zein protein,inducible promoters, such as the light inducible promoter derived fromthe pea rbcS gene (Coruzzi et al., EMBO J. 3: 1671 (1971)) and the actinpromoter from rice (McElroy et al., The Plant Cell. 2: 163-171 (1990)).Seed specific promoters, such as the phaseolin promoter from beans, mayalso be used (Sengupta-Gopalan, Proc. Natl. Acad. Sci. USA. 83:3320-3324 (1985). Other promoters useful in the practice of theinvention are known to those of skill in the art.

The novel tissue specific promoter sequences described here, as well asother promoter sequences, can therefore be employed for the expressionof the transcription factor(s). cDNA clones from a particular tissue canbe isolated and those clones that are expressed specifically in a tissueof interest are identified, for example, using Northern blotting,quantitative PCR and other available methods. In some embodiments, thegene isolated is not present in a high copy number, but is relativelyabundant in specific tissues. The promoter and control elements ofcorresponding genomic clones can then be identified, isolated andutilized using techniques well known to those of skill in the art.

A transcription factor nucleic acid can be combined with a selectedpromoter by standard methods to yield an expression cassette, forexample, as described in Sambrook et al. (MOLECULAR CLONING: ALABORATORY MANUAL. Second Edition (Cold Spring Harbor, N.Y.: Cold SpringHarbor Press (1989); MOLECULAR CLONING: A LABORATORY MANUAL. ThirdEdition (Cold Spring Harbor, N.Y.: Cold Spring Harbor Press (2000)).Briefly, a plasmid containing a promoter such as the 35S CaMV promotercan be constructed as described in Jefferson (Plant Molecular BiologyReporter 5: 387-405 (1987)) or obtained from Clontech Lab in Palo Alto,Calif. (e.g., pBI121 or pBI221). Typically, these plasmids areconstructed to have multiple cloning sites having specificity fordifferent restriction enzymes downstream from the promoter. Thetranscription factor nucleic acids can be subcloned downstream from thepromoter using restriction enzymes and positioned to ensure that thetranscription factor DNA is inserted in proper orientation with respectto the promoter so that the DNA can be expressed. Once the transcriptionfactor nucleic acid is operably linked to a promoter, the expressioncassette so formed can be subcloned into a plasmid or other vector(e.g., an expression vector).

In some embodiments, a cDNA clone encoding a transcription factorprotein is isolated from Arabidopsis. In other embodiments, cDNA clonesfrom other species (that encode a transcription factor protein) areisolated from selected plant tissues, or a nucleic acid encoding amutant or modified transcription factor protein is prepared by availablemethods or as described herein. For example, the nucleic acid encoding amutant or modified transcription factor protein can be any nucleic acidwith a coding region that hybridizes to SEQ ID NO:1, 12, 14, 16, 18, or20, and that can promote expression of a cellulose synthase enzyme.Using restriction endonucleases, the entire coding sequence for thetranscription factor is subcloned downstream of the promoter in a 5′ to3′ sense orientation. The transcription factor protein can be operablylinked to a promoter sequence that is not a nucleic acid segment with asequence that includes SEQ ID NO:3-11, 65-71, or a combination thereof.In other words, while expression of the transcription factor protein canbe self-regulating (e.g., driven by binding of the transcription factorprotein to its own promoter), the expression of the transcription factorprotein can also be controlled by a heterologous promoter that is astrong, weak, inducible, tissue specific, developmentally regulated orsome combination thereof (and that does not include SEQ ID NO:3-11,65-71, or a combination thereof).

Targeting Sequences:

Additionally, expression cassettes can be constructed and employed totarget the transcription factors or other polypeptides of interest tointracellular compartments within plant cells, or to target thetranscription factors or polypeptides of interest for extracellularsecretion.

In general, transcription factors bind to plant chromosomal DNA withinthe nucleus. Therefore, the transcription factor is preferably targetedto the nucleus and not directed to other plant organelles or theextracellular environment. However, there may be instances where is itdesirable to secrete or sequester the transcription factor withinorganelles or storage vesicles (e.g., to facilitate isolation and/orpurification of the transcription factor protein). Similarly,polypeptides of interest can be encoded within expression cassettescontaining one of the cellulose synthase promoters described herein, andit may be desirable to target those polypeptides to variousintracellular compartments or to the extracellular environment.Therefore, the invention contemplates targeting the transcriptionfactor(s) as well as polypeptides of interest to various intracellularand extracellular locations.

A nuclear localization signal or sequence is an amino acid sequencesthat ‘tags’ a protein for import into the cell nucleus by nucleartransport. Transcription factors may naturally have such a nuclearlocalization signal or sequence. Alternatively, a nuclear localizationsignal or sequence can be operably linked to the transcription factorsequence. Transit peptides act by facilitating the transport of proteinsthrough intracellular membranes, e.g., vacuole, vesicle, plastid andmitochondrial membranes, whereas signal peptides direct proteins throughthe extracellular membrane. Polypeptides of interest can be operablylinked to nuclear localization signals/sequences, to transit peptides orto signal peptides.

Targeting to selected intracellular regions can generally be achieved byjoining a DNA sequence encoding a nuclear localization sequence, or atransit peptide or a signal peptide sequence to the coding sequence ofthe transcription factor or the polypeptide of interest. The resultantnuclear localization sequence (or transit, or signal, peptide) willtransport the protein to a particular intracellular (or extracellular)destination. Such sequences (nuclear localization sequences, transitpeptides or signal peptides) may be posttranslationally removed bycellular enzymes. By facilitating transport of the protein intocompartments inside or outside the cell, these sequences can increasethe accumulation of a particular gene product in a particular location.

3′ Sequences:

The expression cassette can also optionally include 3′ nontranslatedplant regulatory DNA sequences that act as a signal to terminatetranscription and allow for the polyadenylation of the resultant mRNA.The 3′ nontranslated regulatory DNA sequence preferably includes fromabout 300 to 1,000 nucleotide base pairs and contains planttranscriptional and translational termination sequences. For example, 3′elements that can be used include those derived from the nopalinesynthase gene of Agrobacterium tumefaciens (Bevan et al., Nucleic AcidResearch. 11:369-385 (1983)), or the terminator sequences for the T7transcript from the octopine synthase gene of Agrobacterium tumefaciens,and/or the 3′ end of the protease inhibitor I or II genes from potato ortomato. Other 3′ elements known to those of skill in the art can also beemployed. These 3′ nontranslated regulatory sequences can be obtained asdescribed in An (Methods in Enzymology, 153:292 (1987)). Many such 3′nontranslated regulatory sequences are already present in plasmidsavailable from commercial sources such as Clontech, Palo Alto, Calif.The 3′ nontranslated regulatory sequences can be operably linked to the3′ terminus of the transcription factor or other polypeptide nucleicacids by standard methods.

Selectable and Screenable Marker Sequences: In order to improveidentification of transformants, a selectable or screenable marker genecan be employed with the expressible transcription factor or otherpolypeptide nucleic acids. “Marker genes” are genes that impart adistinct phenotype to cells expressing the marker gene and thus allowsuch transformed cells to be distinguished from cells that do not havethe marker. Such genes may encode either a selectable or screenablemarker, depending on whether the marker confers a trait which one can‘select’ for the marker by chemical means, i.e., through the use of aselective agent (e.g., a herbicide, antibiotic, or the like), or whethermarker is simply a trait that one can identify through observation ortesting, i.e., by ‘screening’ (e.g., the R-locus trait). Many examplesof suitable marker genes are known to the art and can be employed in thepractice of the invention.

Included within the terms selectable or screenable marker genes are alsogenes which encode a “secretable marker” whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude markers which encode a secretable antigen that can be identifiedby antibody interaction, or secretable enzymes that can be detected bytheir catalytic activity. Secretable proteins fall into a number ofclasses, including small, diffusible proteins detectable, e.g., byELISA; and proteins that are inserted or trapped in the cell wall (e.g.,proteins that include a leader sequence such as that found in theexpression unit of extensin or tobacco PR-S).

With regard to selectable secretable markers, the use of a gene thatencodes a polypeptide that becomes sequestered in the cell wall, wherethe polypeptide includes a unique epitope may be advantageous. Such asecreted antigen marker can employ an epitope sequence that wouldprovide low background in plant tissue, a promoter-leader sequence thatimparts efficient expression and targeting across the plasma membrane,and can produce protein that is bound in the cell wall and yet isaccessible to antibodies. A normally secreted wall protein modified toinclude a unique epitope would satisfy such requirements.

Examples of marker proteins suitable for modification in this mannerinclude extensin or hydroxyproline rich glycoprotein (HPRG). Forexample, the maize IIPRG (Stiefel et al., The Plant Cell. 2: 785-793(1990)) is well characterized in terms of molecular biology, expression,and protein structure and therefore can readily be employed. However,any one of a variety of extensins and/or glycine-rich wall proteins(Keller et al., EMBO J. 8: 1309-1314 (1989)) could be modified by theaddition of an antigenic site to create a screenable marker.

Elements of the present disclosure are exemplified in detail through theuse of particular marker genes. However in light of this disclosure,numerous other possible selectable and/or screenable marker genes willbe apparent to those of skill in the art in addition to the one setforth herein below. Therefore, it will be understood that the followingdiscussion is exemplary rather than exhaustive. In light of thetechniques disclosed herein and the general recombinant techniques thatare known in the art, the present invention readily allows theintroduction of any gene, including marker genes, into a recipient cellto generate a transformed plant cell, e.g., a monocot cell or dicotcell.

Possible selectable markers for use in connection with the presentinvention include, but are not limited to, a neo gene (Potrykus et al.,Mol. Gen. Genet. 199: 183-188 (1985)) which codes for kanamycinresistance and can be selected for using kanamycin, G418, and the like;a bar gene which codes for bialaphos resistance; a gene which encodes analtered EPSP synthase protein (Hinchee et al., Bio/Technology. 6:915-922 (1988)) thus conferring glyphosate resistance; a nitrilase genesuch as barn front Klebsiella ozaenae which confers resistance tobromoxynil (Stalker et at, Science. 242: 419-423 (1988)); a mutantacetolactate synthase gene (ALS) which confers resistance toimidazolinone, sulfonylurea or other ALS-inhibiting chemicals (EuropeanPatent Application 154,204 (1985)); a methotrexate-resistant DHFR gene(Thillet et al., J. Biol. Chem. 263: 12500-12508 (1988)); a dalapondehalogenase gene that confers resistance to the herbicide dalapon; or amutated anthranilate synthase gene that confers resistance to 5-methyltryptophane. Where a mutant EPSP synthase gene is employed, additionalbenefit may be realized through the incorporation of a suitablechloroplast transit peptide, CTP (European Patent Application 0 218 571(1987)).

Another selectable marker gene capable of being used in for selection oftransformants is the gene that encode the enzyme phosphinothricinacetyltransferase, such as the bar gene from Streptomyces hygroscopicusor the pat gene from Streptomyces viridochromogenes (U.S. Pat. No.5,550,318). The enzyme phosphinothricin acetyl transferase (PAT)inactivates the active ingredient in the herbicide bialaphos,phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami etal., Mol. Gen. Genet. 205: 42-50 (1986); Twell et al., Plant Physiol.91: 1270-1274 (1989)) causing rapid accumulation of ammonia and celldeath. The success in using this selective system in conjunction withmonocots was surprising because of the major difficulties that have beenreported in transformation of cereals (Potrykus, Trends Biotech.7:269-273 (1989)).

Screenable markers that may be employed include, but are not limited to,a β-glucuronidase or uidA gene (GUS) that encodes an enzyme for whichvarious chromogenic substrates are known; an R-locus gene, which encodesa product that regulates the production of anthocyanin pigments (redcolor) in plant tissues (Dellaporta et al., In: Chromosome Structure andFunction: Impact of New Concepts, 18^(th) Stadler Genetics Symposium, J.P. Gustafson and R. Appels, eds. (New York: Plenum Press) pp. 263-282(1988)); a β-lactamase gene (Sutcliffe, Proc. Natl. Acad. Sci. USA. 75:3737-3741 (1978)), which encodes an enzyme for which various chromogenicsubstrates are known (e.g., PADAC, a chromogenic cephalosporin); a xylEgene (Zukowsky et al., Proc. Natl. Acad. Sci. USA. 80:1101 (1983)) whichencodes a catechol dioxygenase that can convert chromogenic catechols;an α-amylase gene (Ikuta et al., Bio/technology 8: 241-242 (1990)); atyrosinase gene (Katz et al., J. Gen. Microbiol. 129: 2703-2714 (1983))which encodes an enzyme capable of oxidizing tyrosine to DOPA anddopaquinone which in turn condenses to form the easily detectablecompound melanin; a β-galactosidase gene, which encodes an enzyme forwhich there are chromogenic substrates; a luciferase (lux) gene (Ow etal., Science. 234: 856-859, 1986), which allows for bioluminescencedetection; or an aequorin gene (Prasher et al., Biochem. Biophys. Res.Comm. 126: 1259-1268 (1985)), which may be employed in calcium-sensitivebioluminescence detection, or a green or yellow fluorescent protein gene(Niedz et al., Plant Cell Reports. 14: 403 (1995).

For example, genes from the maize R gene complex can be used asscreenable markers. The R gene complex in maize encodes a protein thatacts to regulate the production of anthocyanin pigments in most seed andplant tissue. Maize strains can have one, or as many as four, R allelesthat combine to regulate pigmentation in a developmental and tissuespecific manner. A gene froth the R gene complex does not harm thetransformed cells. Thus, an R gene introduced into such cells will causethe expression of a red pigment and, if stably incorporated, can bevisually scored as a red sector. If a maize line carries dominantalleles for genes encoding the enzymatic intermediates in theanthocyanin biosynthetic pathway (C2, A1, A2, Bz1 and Bz2), but carriesa recessive allele at the R locus, transformation of any cell from thatline with R will result in red pigment formation. Exemplary linesinclude Wisconsin 22 that contains the rg-Stadler allele and TR112, aK55 derivative that is r-g, b, P1. Alternatively any genotype of maizecan be utilized if the C1 and R alleles are introduced together.

The R gene regulatory regions may be employed in chimeric constructs inorder to provide mechanisms for controlling the expression of chimericgenes. More diversity of phenotypic expression is known at the R locusthan at any other locus (Coe et al., in Corn and Corn Improvement, eds.Sprague, G. F. & Dudley, J. W. (Am. Soc. Agron., Madison, Wis.), pp.81-258 (1988)). It is contemplated that regulatory regions obtained fromregions 5′ to the structural R gene can be useful in directing theexpression of genes, e.g., insect resistance, drought resistance,herbicide tolerance or other protein coding regions. For the purposes ofthe present invention, it is believed that any of the various R genefamily members may be successfully employed (e.g., P, S, Lc, etc.).However, one that can be used is Sn (particularly Sn:bol3). Sn is adominant member of the R gene complex and is functionally similar to theR and B loci in that Sn controls the tissue specific deposition ofanthocyanin pigments in certain seedling and plant cells, therefore, itsphenotype is similar to R.

A further screenable marker contemplated for use in the presentinvention is firefly luciferase, encoded by the lux gene. The presenceof the lux gene in transformed cells may be detected using, for example,X-ray film, scintillation counting, fluorescent spectrophotometry,low-light video cameras, photon counting cameras or multiwellluminometry. It is also envisioned that this system may be developed forpopulation screening for bioluminescence, such as on tissue cultureplates, or even for whole plant screening.

Other Optional Sequences:

An expression cassette of the invention can also further compriseplasmid DNA. Plasmid vectors include additional DNA sequences thatprovide for easy selection, amplification, and transformation of theexpression cassette in prokaryotic and eukaryotic cells, e.g.,pUC-derived vectors such as pUC8, pUC9, pUC18, pUC19, pUC23, pUC119, andpUC120, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors,or pBS-derived vectors. The additional DNA sequences include origins ofreplication to provide for autonomous replication of the vector,additional selectable marker genes (e.g., antibiotic or herbicideresistance), unique multiple cloning sites providing for multiple sitesto insert DNA sequences or genes encoded in the expression cassette andsequences that enhance transformation of prokaryotic and eukaryotic,cells.

Another vector that is useful for expression in both plant andprokaryotic cells is the binary Ti plasmid (as disclosed in Schilperoortet al., U.S. Pat. No. 4,940,838) as exemplified by vector pGA582. Thisbinary Ti plasmid vector has been previously characterized by An(Methods in Enzymology, 153: 292 (1987)) and is available from Dr. An.This binary Ti vector can be replicated in prokaryotic bacteria such asE. coli and Agrobacterium. The Agrobacterium plasmid vectors can be usedto transfer the expression cassette to dicot plant cells, and undercertain conditions to monocot cells, such as rice cells. The binary Tivectors preferably include the nopaline T DNA right and left borders toprovide for efficient plant cell transformation, a selectable markergene, unique multiple cloning sites in the T border regions, the colE1replication of origin and a wide host range replicon. The binary Tivectors carrying an expression cassette of the invention can be used totransform both prokaryotic and eukaryotic cells, but is preferably usedto transform dicot plant cells.

In Vitro Screening of Expression Cassettes:

Once the expression cassette is constructed and subcloned into asuitable plasmid, it can be screened for the ability to express thetranscription factor or another polypeptide of interest. For example, anexpression cassette encoding a transcription factor can be screened toascertain whether it can promote expression of a cellulose synthase bymethods described herein or other available methods for detectingcellulose. An expression cassette encoding a other polypeptides ofinterest (with a promoter that includes a segment with a sequence suchas any of SEQ ID NOs: 3-11, 65-71, or a combination thereof) can bescreened to ascertain whether it can promote expression of thepolypeptide, for example, by immunological detection of the polypeptideof interest, by detection of the activity of the polypeptide, byhybridization or PCR detection of transcripts encoding the polypeptide,or by other procedures available to those of skill in the art.

DNA Delivery of the DNA Molecules into Host Cells: Nucleic acidsencoding a transcription factor or another polypeptide can be introducedinto host cells by a variety of methods. For example, a preselected cDNAencoding the selected transcription factor or other polypeptide can beintroduced into a recipient cell to create a transformed cell byavailable procedures. The frequency of occurrence of cells taking upexogenous (foreign) DNA may be low. Moreover, it is most likely that notall recipient cells receiving DNA segments or sequences will result in atransformed cell wherein the DNA is stably integrated into the plantgenome and/or expressed. Some may show only initial and transient geneexpression. However, certain cells from virtually any dicot or monocotspecies may be stably transformed, and these cells can be regeneratedinto transgenic plants, through the application of the techniquesdisclosed herein.

Another aspect of the invention is an isolated plant or plant cell thathas one of the transcription factors or promoters described hereinintroduced into the plant or cell, e.g., as a nucleic acid encoding thetranscription factor or promoter. The isolated plant or plant cell canalso have any of the isolated transcription factors described herein asa protein product. The plant can be a monocotyledon or a dicotyledon.Another aspect of the invention includes plant cells (e.g., embryoniccells or other cell lines) that can regenerate fertile transgenic plantsand/or seeds. The cells can be derived from either monocotyledons ordicotyledons. Suitable examples of plant species include wheat, rice,Arabidopsis, tobacco, maize, soybean, poplar, and the like. In someembodiments, the plant or cell is a monocotyledon plant or cell. Forexample, the plant or cell can be a maize plant or cell. The cell(s) maybe in a suspension cell culture or may be in an intact plant part, suchas an immature embryo, or in a specialized plant tissue, such as callus,such as Type I or Type II callus.

Plants or plant cells that can have one of the transcription factors orpromoters described herein introduced therein include but are notlimited to grass species, oil and/or starch plants (canola, potatoes,lupins, sunflower and cottonseed), forage plants (alfalfa, clover andfescue), grains (maize, wheat, barley, oats, rice, sorghum, millet andrye), grasses (switchgrass, prairie grass, wheat grass, sudangrass,sorghum, straw-producing plants), softwood, hardwood and other woodyplants (e.g., those used for paper production such as poplar species,pine species, and eucalyptus). In some embodiments the plant is agymnosperm. Examples of plants useful for pulp and paper productioninclude most pine species such as loblolly pine, Jack pine, Southernpine, Radiata pine, spruce, Douglas fir and others. Hardwoods that canbe modified as described herein include aspen, poplar, eucalyptus, andothers. Plants useful for making biofuels and ethanol include corn,grasses (e.g., miscanthus, switchgrass, and the like), as well as treessuch as poplar, aspen, willow, and the like. Plants useful forgenerating dairy forage include legumes such as alfalfa, as well asforage grasses such as bromegrass, and bluestem.

Transformation of the cells of the plant tissue source can be conductedby any one of a number of methods known to those of skill in the art.Examples are: Transformation by direct DNA transfer into plant cells byelectroporation (U.S. Pat. No. 5,384,253 and U.S. Pat. No. 5,472,869,Dekeyser et al., The Plant Cell. 2: 591-602 (1990)); direct DNA transferto plant cells by PEG precipitation (Hayashimoto et al., Plant Physiol.93: 857-863 (1990)); direct DNA transfer to plant cells bymicroprojectile bombardment (McCabe et al., Bio/Technology. 6: 923-926(1988); Gordon-Kamm et al., The Plant Cell. 2: 603-618 (1990); U.S. Pat.Nos. 5,489,520; 5,538,877; and 5,538,880) and DNA transfer to plantcells via infection with Agrobacterium. Methods such as microprojectilebombardment or electroporation can be carried out with “naked” DNA wherethe expression cassette may be simply carried on any E. coli-derivedplasmid cloning vector. In the case of viral vectors, it is desirablethat the system retain replication functions, but lack functions fordisease induction.

One method for dicot transformation, for example, involves infection ofplant cells with Agrobacterium tumefaciens using the leaf-disk protocol(Horsch et al., Science 227: 1229-1231 (1985). Monocots such as Zea mayscan be transformed via microprojectile bombardment of embryogenic callustissue or immature embryos, or by electroporation following partialenzymatic degradation of the cell wall with a pectinase-containingenzyme (U.S. Pat. Nos. 5,384,253; and 5,472,869). For example,embryogenic cell lines derived from immature Zea mays embryos can betransformed by accelerated particle treatment as described byGordon-Kamm et al. (The Plant Cell. 2: 603-618 (1990)) or U.S. Pat. Nos.5,489,520; 5,538,877 and 5,538,880, cited above. Excised immatureembryos can also be used as the target for transformation prior totissue culture induction, selection and regeneration as described inU.S. application Ser. No. 08/112,245 and PCT publication WO 95/06128.Furthermore, methods for transformation of monocotyledonous plantsutilizing Agrobacterium tumefaciens have been described by Hiei et at(European Patent 0 604 662, 1994) and Saito et al. (European Patent 0672 752, 1995).

Methods such as microprojectile bombardment or electroporation arecarried out with “naked” DNA where the expression cassette may be simplycarried on any E. coli-derived plasmid cloning vector. In the case ofviral vectors, it is desirable that the vectors retain replicationfunctions, but not have functions for disease induction.

The choice of plant tissue source for transformation will depend on thenature of the host plant and the transformation protocol. Useful tissuesources include callus, suspension culture cells, protoplasts, leafsegments, stem segments, tassels, pollen, embryos, hypocotyls, tubersegments, meristematic regions, and the like. The tissue source isselected and transformed so that it retains the ability to regeneratewhole, fertile plants following transformation, i.e., containstotipotent cells. Type I or Type II embryonic maize callus and immatureembryos are preferred Zea mays tissue sources. Selection of tissuesources for transformation of monocots is described in detail in U.S.application Ser. No. 08/112,245 and PCT publication WO 95/06128.

The transformation is carried out under conditions directed to the planttissue of choice. The plant cells or tissue are exposed to the DNA orRNA carrying the transcription factor nucleic acids for an effectiveperiod of time. This may range from a less than one second pulse ofelectricity for electroporation to a 2-3 day co-cultivation in thepresence of plasmid-bearing Agrobacterium cells. Buffers and media usedwill also vary with the plant tissue source and transformation protocol.Many transformation protocols employ a feeder layer of suspended culturecells (tobacco or Black Mexican Sweet corn, for example) on the surfaceof solid media plates, separated by a sterile filter paper disk from theplant cells or tissues being transformed.

Electroporation:

Where one wishes to introduce DNA by means of electroporation, it iscontemplated that the method of Krzyzek et al. (U.S. Pat. No. 5,384,253)may be advantageous. In this method, certain cell wall-degradingenzymes, such as pectin-degrading enzymes, are employed to render thetarget recipient cells more susceptible to transformation byelectroporation than untreated cells. Alternatively, recipient cells canbe made more susceptible to transformation, by mechanical wounding.

To effect transformation by electroporation, one may employ eitherfriable tissues such as a suspension cell cultures, or embryogeniccallus, or alternatively, one may transform immature embryos or otherorganized tissues directly. The cell walls of the preselected cells ororgans can be partially degraded by exposing them to pectin-degradingenzymes (pectinases or pectolyases) or mechanically wounding them in acontrolled manner. Such cells would then be receptive to DNA uptake byelectroporation, which may be carried out at this stage, and transformedcells then identified by a suitable selection or screening protocoldependent on the nature of the newly incorporated DNA.

Microprojectile Bombardment:

A further advantageous method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method,microparticles may be coated with DNA and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like.

It is contemplated that in some instances DNA precipitation onto metalparticles would not be necessary for DNA delivery to a recipient cellusing microprojectile bombardment. For example, non-embryogenic BlackMexican Sweet maize cells can be bombarded with intact cells of thebacteria E. coli or Agrobacterium tumefaciens containing plasmids witheither the β-glucuronidase or bar gene engineered for expression inmaize. Bacteria can be inactivated by ethanol dehydration prior tobombardment. A low level of transient expression of the β-glucuronidasegene may be observed 24-48 hours following DNA delivery. In addition,stable transformants containing the bar gene can be recovered followingbombardment with either E. coli or Agrobacterium tumefaciens cells. Itis contemplated that particles may contain DNA rather than be coatedwith DNA. Hence it is proposed that particles may increase the level ofDNA delivery but are not, in and of themselves, necessary to introduceDNA into plant cells.

An advantage of microprojectile bombardment, in addition to it being aneffective means of reproducibly stably transforming monocots, is thatthe isolation of protoplasts (Christou et al., PNAS. 84: 3962-3966(1987)), the formation of partially degraded cells, or thesusceptibility to Agrobacterium infection is not required. Anillustrative embodiment of a method for delivering DNA into maize cellsby acceleration is a Biolistics Particle Delivery System, which can beused to propel particles coated with DNA or cells through a screen, suchas a stainless steel or Nytex screen, onto a filter surface covered withmaize cells cultured in suspension (Gordon-Kamm et al., The Plant Cell.2:603-618 (1990)). The screen disperses the particles so that they arenot delivered to the recipient cells in large aggregates. It is believedthat a screen intervening between the projectile apparatus and the cellsto be bombarded reduces the size of projectile aggregate and maycontribute to a higher frequency of transformation, by reducing damageinflicted on the recipient cells by an aggregated projectile.

For bombardment, cells in suspension are preferably concentrated onfilters or solid culture medium. Alternatively, immature embryos orother target cells may be arranged on solid culture medium. The cells tobe bombarded are positioned at an appropriate distance below themacroprojectile stopping plate. If desired, one or more screens are alsopositioned between the acceleration device and the cells to bebombarded. Through the use of such techniques one may obtain up to 1000or more foci of cells transiently expressing a marker gene. The numberof cells in a focus which express the exogenous gene product 48 hourspost-bombardment often range from about 1 to 10 and average about 1 to3.

In bombardment transformation, one may optimize the prebombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment can influence transformation frequency.Physical factors are those that involve manipulating theDNA/microprojectile precipitate or those that affect the path andvelocity of either the macro- or microprojectiles. Biological factorsinclude all steps involved in manipulation of cells before andimmediately after bombardment, the osmotic adjustment of target cells tohelp alleviate the trauma associated with bombardment, and also thenature of the transforming DNA, such as linearized DNA or intactsupercoiled plasmid DNA.

One may wish to adjust various bombardment parameters in small scalestudies to fully optimize the conditions and/or to adjust physicalparameters such as gap distance, flight distance, tissue distance, andhelium pressure. One may also minimize the trauma reduction factors(TRFs) by modifying conditions which influence the physiological stateof the recipient cells and which may therefore influence transformationand integration efficiencies. For example, the osmotic state, tissuehydration and the subculture stage or cell cycle of the recipient cellsmay be adjusted for optimum transformation. Execution of such routineadjustments will be known to those of skill in the art.

After effecting delivery of a transcription factor nucleic acid (orother nucleic acid encoding a desirable polypeptide) to recipient cellsby any of the methods discussed above, the transformed cells can beidentified for further culturing and plant regeneration. As mentionedabove, in order to improve the ability to identify transformants, onemay employ a selectable or screenable marker gene as, or in addition to,the expressible transcription factor nucleic acids. In this case, onewould then generally assay the potentially transformed cell populationby exposing the cells to a selective agent or agents, or one wouldscreen the cells for the desired marker gene trait.

Selection:

An exemplary embodiment of methods for identifying transformed cellsinvolves exposing the bombarded cultures to a selective agent, such as ametabolic inhibitor, an antibiotic, herbicide or the like. Cells thathave been transformed and have stably integrated a marker geneconferring resistance to the selective agent used, will grow and dividein culture. Sensitive cells will not be amenable to further culturing.

To use the bar-bialaphos or the EPSPS-glyphosate selective system,bombarded tissue is cultured for about 0-28 days on nonselective mediumand subsequently transferred to medium containing from about 1-3 mg/lbialaphos or about 1-3 mM glyphosate, as appropriate. While ranges ofabout 1-3 mg/l bialaphos or about 1-3 mM glyphosate can be employed, itis proposed that ranges of at least about. 0.1-50 mg/l bialaphos or atleast about 0.1-50 mM glyphosate will find utility in the practice ofthe invention. Tissue can be placed on any porous, inert, solid orsemi-solid support for bombardment, including but not limited to filtersand solid culture medium. Bialaphos and glyphosate are provided asexamples of agents suitable for selection of transformants, but thetechnique of this invention is not limited to them.

An example of a screenable marker trait is the red pigment producedunder the control of the R-locus in maize. This pigment may be detectedby culturing cells on a solid support containing nutrient media capableof supporting growth at this stage and selecting cells front colonies(visible aggregates of cells) that are pigmented. These cells may becultured further, either in suspension or on solid media. The R-locus isuseful for selection of transformants from bombarded immature embryos.In a similar fashion, the introduction of the C1 and B genes will resultin pigmented cells and/or tissues.

The enzyme luciferase is also useful as a screenable marker in thecontext of the present invention. In the presence of the substrateluciferin, cells expressing luciferase emit light which can be detectedon photographic or X-ray film, in a luminometer (or liquid scintillationcounter), by devices that enhance night vision, or by a highly lightsensitive video camera, such as a photon counting camera. All of theseassays are nondestructive and transformed cells may be cultured furtherfollowing identification. The photon counting camera is especiallyvaluable as it allows one to identify specific cells or groups of cellswhich are expressing luciferase and manipulate those in real time.

It is further contemplated that combinations of screenable andselectable markers may be useful for identification of transformedcells. For example, selection with a growth inhibiting compound, such asbialaphos or glyphosate at concentrations below those that cause 100%inhibition followed by screening of growing tissue for expression of ascreenable marker gene such as luciferase would allow one to recovertransformants from cell or tissue types that are not amenable toselection alone. In an illustrative embodiment embryogenic Type IIcallus of Zea mays L. can be selected with sublethal levels ofbialaphos. Slowly growing tissue was subsequently screened forexpression of the luciferase gene and transformants can be identified.

Regeneration and Seed Production:

Cells that survive the exposure to the selective agent, or cells thathave been scored positive in a screening assay, are cultured in mediathat supports regeneration of plants. One example of a growth regulatorthat can be used for such purposes is dicamba or 2,4-D. However, othergrowth regulators may be employed, including NAA, NAA+2,4-D or perhapseven picloram. Media improvement in these and like ways can facilitatethe growth of cells at specific developmental stages. Tissue can bemaintained on a basic media with growth regulators until sufficienttissue is available to begin plant regeneration efforts, or followingrepeated rounds of manual selection, until the morphology of the tissueis suitable for regeneration, at least two weeks, then transferred tomedia conducive to maturation of embryoids. Cultures are typicallytransferred every two weeks on this medium. Shoot development signalsthe time to transfer to medium lacking growth regulators.

The transformed cells, identified by selection or screening and culturedin an appropriate medium that supports regeneration, can then be allowedto mature into plants. Developing plantlets are transferred to soil-lessplant growth mix, and hardened, e.g., in an environmentally controlledchamber at about 85% relative humidity, about 600 ppm CO₂, and at about25-250 microeinsteins/sec·m² of light. Plants can be matured either in agrowth chamber or greenhouse. Plants are regenerated from about 6 weeksto 10 months after a transformant is identified, depending on theinitial tissue. During regeneration, cells are grown on solid media intissue culture vessels. Illustrative embodiments of such vessels arepetri dishes and Plant Con™. Regenerating plants can be grown at about19° C. to 28° C. After the regenerating plants have reached the stage ofshoot and root development, they may be transferred to a greenhouse forfurther growth and testing.

Mature plants are then obtained from cell lines that are known toexpress the trait. In some embodiments, the regenerated plants are selfpollinated. In addition, pollen obtained from the regenerated plants canbe crossed to seed grown plants of agronomically important inbred lines.In some cases, pollen front plants of these inbred lines is used topollinate regenerated plants. The trait is genetically characterized byevaluating the segregation of the trait in first and later generationprogeny. The heritability and expression in plants of traits selected intissue culture are of particular importance if the traits are to becommercially useful.

Regenerated plants can be repeatedly crossed to inbred plants in orderto introgress the transcription factor nucleic acids into the genome ofthe inbred plants. This process is referred to as backcross conversion.When a sufficient number of crosses to the recurrent inbred parent havebeen completed in order to produce a product of the backcross conversionprocess that is substantially isogenic with the recurrent inbred parentexcept for the presence of the introduced transcription factor or otherpromoter-polypeptide encoding nucleic acids, the plant isself-pollinated at least once in order to produce a homozygous backcrossconverted inbred containing the transcription factor or otherpromoter-polypeptide nucleic acids. Progeny of these plants are truebreeding.

Alternatively, seed from transformed monocot plants regenerated fromtransformed tissue cultures is grown in the field and self-pollinated togenerate true breeding plants.

Seed from the fertile transgenic plants can then be evaluated for thepresence and/or expression of the transcription factor or otherpolypeptide nucleic acids (or the encoded transcription factor or otherpolypeptide). Transgenic plant and/or seed tissue can be analyzed fortranscription factor expression using standard methods such as SDSpolyacrylamide gel electrophoresis, liquid chromatography (e.g., HPLC)or other means of detecting a product of transcription factor activity(e.g., increased cellulose or heightened expression of a cellulosesynthase) or a product of the polypeptide of interest.

Once a transgenic seed expressing the transcription factor or otherpolypeptide sequence is identified, the seed can be used to develop truebreeding plants. The true breeding plants are used to develop a line ofplants that express the transcription factor, contain one of thecellulose synthase promoters described herein and/or contain a nucleicacid encoding such a promoter linked to a polypeptide of interest, whilestill maintaining other desirable functional agronomic traits. Addingthe trait of increased transcription factor or other polypeptideexpression to the plant can be accomplished by back-crossing with thistrait with plants that do not exhibit this trait and by studying thepattern of inheritance in segregating generations. Those plantsexpressing the target trait in a dominant fashion are preferablyselected. Back-crossing is carried out by crossing the original fertiletransgenic plants with a plant from an inbred line exhibiting desirablefunctional agronomic characteristics while not necessarily expressingthe trait of expression of a transcription factor and/or other desiredpolypeptide in the plant. The resulting progeny are then crossed back tothe parent that expresses the trait. The progeny from this cross willalso segregate so that some of the progeny carry the trait and some donot. This back-crossing is repeated until an inbred line with thedesirable functional agronomic trails, and with expression of thedesired trait within the plant. Such expression of the increasedexpression of the transcription factor or other polypeptide in plant canbe expressed in a dominant fashion.

Subsequent to back-crossing, the new transgenic plants can be evaluatedfor expression of the transcription factor or other polypeptide. Forexample, when the transcription factor is expressed the weight percentof cellulose within the plant or within selected tissues of the plant isincreased. Detection of increased cellulose can be done, for example, bystaining plant tissues for cellulose or by observing whether the tensilestrength of plant fibers is increased or otherwise modulated relative toa plant that does not contain the exogenously added transcriptionfactor. The new transgenic plants can also be evaluated for a battery offunctional agronomic characteristics such as lodging, kernel hardness,yield, resistance to disease and insect pests, drought resistance,and/or herbicide resistance.

Plants that may be improved by these methods include but are not limitedto fiber-containing plants, trees, flax, grains (maize, wheat, barley,oats, rice, sorghum, millet and rye), grasses (switchgrass, prairiegrass, wheat grass, sudangrass, sorghum, straw-producing plants),softwood, hardwood and other woody plants (e.g., those used for paperproduction such as poplar species, pine species, and eucalyptus), oiland/or starch plants (canola, potatoes, lupins, sunflower andcottonseed), and forage plants (alfalfa, clover and fescue). In someembodiments the plant is a gymnosperm Examples of plants useful for pulpand paper production include most pine species such as loblolly pine,Jack pine, Southern pine, Radiata pine, spruce, Douglas fir and others.Hardwoods that can be modified as described herein include aspen,poplar, eucalyptus, and others. Plants useful for making biofuels andethanol include corn, grasses (e.g., miscanthus, switchgrass, and thelike), as well as trees such as poplar, aspen, willow, and the like.Plants useful for generating dairy forage include legumes such asalfalfa, as well as forage grasses such as bromegrass, and bluestem.

Determination of Stably Transformed Plant Tissues:

To confirm the presence of the transcription factor or otherpromoter-polypeptide-encoding nucleic acids in the regenerating plants,or in seeds or progeny derived from the regenerated plant, a variety ofassays may be performed. Such assays include, for example, molecularbiological assays available to those of skill in the art, such asSouthern and Northern blotting, and PCR; biochemical assays, such asdetecting the presence of a protein product, e.g., by immunologicalmeans (ELISAs and Western blots) or by enzymatic function; plant partassays, such as leaf, seed or root assays; and also, by analyzing thephenotype of the whole regenerated plant.

Whereas DNA analysis techniques may be conducted using DNA isolated fromany part of a plant, RNA may only be expressed in particular cells ortissue types and so RNA for analysis can be obtained from those tissues.PCR techniques may also be used for detection and quantification of RNAproduced from introduced transcription factor nucleic acids. PCR also beused to reverse transcribe RNA into DNA, using enzymes such as reversetranscriptase, and then this DNA can be amplified through the use ofconventional PCR techniques. Further information about the nature of theRNA product may be obtained by Northern blotting. This technique willdemonstrate the presence of an RNA species and give information aboutthe integrity of that RNA. The presence or absence of an RNA species canalso be determined using dot or slot blot Northern hybridizations. Thesetechniques are modifications of Northern blotting and also demonstratethe presence or absence of an RNA species.

While Southern blotting and PCR may be used to detect the transcriptionfactor nucleic acid in question, they do not provide information as towhether the preselected DNA segment is being expressed. Expression maybe evaluated by specifically identifying the protein products of theintroduced transcription factor nucleic acids or evaluating thephenotypic changes brought about by their expression.

Assays for the production and identification of specific proteins maymake use of physical-chemical, structural, functional, or otherproperties of the proteins. Unique physical-chemical or structuralproperties allow the proteins to be separated and identified byelectrophoretic procedures, such as native or denaturing gelelectrophoresis or isoelectric focusing, or by chromatographictechniques such as ion exchange, liquid chromatography or gel exclusionchromatography. The unique structures of individual proteins offeropportunities for use of specific antibodies to detect their presence informats such as an ELISA assay. Combinations of approaches may beemployed with even greater specificity such as Western blotting in whichantibodies are used to locate individual gene products that have beenseparated by electrophoretic techniques. Additional techniques may beemployed to absolutely confirm the identity of the transcription factoror other polypeptide such as evaluation by amino acid sequencingfollowing purification. The Examples of this application also provideassay procedures for detecting and quantifying transcription factor orother polypeptide or enzyme activities. Other procedures may beadditionally used.

The expression of a gene product can also be determined by evaluatingthe phenotypic results of its expression. These assays also may takemany forms including but not limited to analyzing changes in thechemical composition, morphology, or physiological properties of theplant.

Definitions

As used herein, “isolated” means a nucleic acid or polypeptide has beenremoved from its natural or native cell. Thus, the nucleic acid orpolypeptide can be physically isolated from the cell, or the nucleicacid or polypeptide can be present or maintained in another cell whereit is not naturally present or synthesized.

As used herein, a “native” nucleic acid or polypeptide means a DNA, RNAor amino acid sequence or segment that has not been manipulated invitro, i.e., has not been isolated, purified, and/or amplified.

As used herein, “natural promoter” means a nucleic acid segment withpromoter function that is naturally operably linked to a coding regionin the native genome of an organism (e.g., a plant). For example, anatural promoter for a CESA gene is the promoter that is present in thenative genome of a plant species.

As used herein, “transgene” means a recombinantly engineered nucleicacid that includes at least a promoter segment that is operably linkedto a segment encoding an amino acid sequence. The promoter can be (butneed not be) heterologous to the segment encoding an amino acidsequence.

The following non-limiting Examples illustrate how aspects of theinvention have been developed and can be made and used.

Example 1: Materials and Methods

This Example describes some of the materials and methods used indevelopment of the invention.

Plant Materials and Growth Conditions

Arabidopsis thaliana, ecotype Columbia (Col-0), was used in both thewild-type and transgenic experiments. Plants were grown on soil in agrowth chamber (16 h light/8 h dark) at 23° C.

RNA Extraction and Quantitative Real-Time PCR

Total RNA was extracted from liquid nitrogen-frozen samples using PlantRNeasy extraction kit (Qiagen). For quantitative real-time PCR analysis,total RNA was treated with DNase I and used for first-strand cDNAsynthesis by SuperScript II Reverse Transcriptase (Invitrogen).Real-time PCR was performed using SYBR Premix Ex Taq™ (Takara) and ABIPrism 7900HT Sequence Detection System (ABI). The relative mRNA levelswere determined by normalizing the PCR threshold cycle number of eachgene with that of the ACT8 reference gene. Three biological replicateswere used in the experiments.

Protein Expression and Purification

MYB46 was fused in frame with GST and expressed in Escherichia colistrain Rosetta gami (Novagen). The expression of the recombinantGST-MYB46 protein was induced by culturing the E. coli cells for 16 h at16° C. in LB medium supplemented with 0.3 mM IPTG (isopropylβ-D-thiogalactopyranoside). The recombinant proteins for electrophoreticmobility shift assays (EMSAs) were purified using MagneGST™ ProteinPurification System (Promega) according to the protocol provided in thekit.

Electrophoretic Mobility Shift Assay (EMSA)

DNA fragments for EMSA were obtained by PCR-amplification and labeledwith [γ-³²P]ATP using T4 polynucleotide kinase (NEB). The end-labeledprobes were purified with Microspin S-200 HR column (GE Healthcare). Thelabeled DNA fragments were incubated for 25 min with 50 ng of GST-MYB46in a binding buffer [10 mM. Tris (pH 7.5), 50 mM KCl, 1 mM DTT, 2.5%glycerol, 5 mM MgCl₂, 100 μg/ml BSA, and 50 ng/μL poly(dI-dC)]. Fivepercent polyacrylamide gel electrophoresis (PAGE) was used to separatethe recombinant protein-bound DNA fragments from the unbound ones. Thegel was dried and placed in a film cassette and exposed to X-ray film(Kodak) for overnight. Radioactive fragments were visualized byautoradiography.

Dexamethasone Inducible Activation System for Confirmation of DirectTargets.

The full-length cDNA of MYB46 was fused to the N terminus of theglucocorticoid receptor (GR) coding sequence and ligated between theCaMV 35S promoter and the nopaline synthase terminator in pTrGUS vector(Ko et al., 2009). The MYB46-GR expression construct was introduced intoArabidopsis leaf protoplasts alone or together with the AtC3H14 promoterGUS construct (Ko et al., 2009). The primers used for the PCRamplification of the full-length MYB46 glucocorticoid receptor andAtC3H14 promoter were shown in Table S1. Preparation of Arabidopsis leafprotoplasts and transfection were carried out as described previously(Ko et al., 2009; Sheen, 2001). To activate MYB46, the protoplasts weretreated with 10 μM dexamethasone (DEX, Sigma) for 5 h. The controlprotoplasts were mock-treated with the same concentration (0.01%) ofethanol used to dissolve DEX. To inhibit new protein synthesis, theprotein synthesis inhibitor cycloheximide (2 μM) was added 30 min beforeaddition of DEX (Zhong et al. 2008). After the treatments, theprotoplasts were harvested for quantitative real-time PCR analysis andGUS activity analysis (Ko et al., 2009). The expression level of eachgene in the control protoplasts without DEX treatment was set to 1, andthree biological replications were used in the experiments.

Chromatin Immunoprecipitation Analysis

The full-length cDNA of MYB46 was fused in frame with GFP and ligateddown-stream of the GAL4 upstream activation sequence in pTA7002 binaryvector (Aoyama and Chua, 1997). The vector construct was used in theAgrobacterium-mediated transformation of Arabidopsis thaliana (Col-0)plants.

The MYB46-GFP/pTA7002 transgenic plants were grown on soil for threeweeks before the DEX treatment. DEX (10 μM) was applied by spraying with0.02% silwet surfactant (Lehle Seeds). Eight hours after the DEXtreatment, aboveground portion of the plants were harvested andcross-linked with 1% formaldehyde for 10 min under vacuum. Thecross-linking was quenched in 0.125 M glycine for 5 min. Thecross-linked samples were washed twice with deionized water and thenground in liquid nitrogen into a fine powder for extraction ofchromatin. To extract chromatin, 2 g of the ground powder wasresuspended in 30 ml of Extraction Buffer 1 [10 mM Tris-HCl (pH 8.0),0.4 M sucrose, 5 mM 2-mercaptoethanol, 1 mM PMSF, 1 tablet/50 mlprotease inhibitor cocktail, and 4 μg/ml pepstain A] and filteredthrough two layers of Miracloth before centrifugation at 2500 g for 20min at 4° C. The pellet was resuspended in 1 ml of Extraction Buffer 2[10 mM Tris-HCl (pH 8.0), 0.25 M sucrose, 10 mM MgCl₂, 1% Triton X-100,5 mM 2-mercaptoethanol, 1 mM. PMSF, 1× protease inhibitor cocktail, and4 μg/ml pepstain A] and centrifuged at 14,000 g for 10 min at 4° C. Thepellet was resuspended in 300 μl of Extraction Buffer 3 [10 mM Tris-HCl(pH 8.0), 1.7 M sucrose, 0.15% Triton X-100, 2 mM MgCl₂, 5 mM2-mercaptoethanol, 1 mM PMSF, 1× protease inhibitor cocktail, and 4μg/ml pepstain A] and then layered on top of a cushion of 300 μl ofExtraction Buffer 3 and centrifuged at 14,000 g for 1 h at 4° C. Thechromatin pellet was resuspended in 500 μl of ice cold Nuclei LysisBuffer [50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1% SDS, 1 mM PMSF, 1×protease inhibitor cocktail, and 4 μg/ml pepstain A] and sonicated tosmall fragments with an average fragment size of 600-800 bp. Thesonicated chromatin was diluted 10 times in ChIP Dilution Buffer [16.7mMTris-HCl (pH 8.0), 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl, 1 mMPMSF, 1× protease inhibitor cocktail, and 4 μg/ml pepstain A] andprecleared by incubation with Protein A agarose beads (Roch AppliedScience) for 1 h at 4° C. The precleared chromatin was then incubatedwith 2 μg of GFP antibody (Abcam) overnight at 4° C. The MYB46-GFP-boundchromatin was purified by incubation with Protein A agarose beads for 1h at 4° C. The agarose beads was washed sequentially with 1 ml each ofthe following wash buffers by gently rocking on a shaker for 5 min at 4°C.: (1) Low-Salt Wash Buffer [20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.2%SDS, 0.5% Triton X-100 and 2 mM EDTA], (2) High-Salt Wash Buffer [20 mMTris-HCl (pH 8.0), 500 mM NaCl, 0.2% SDS, 0.5% Triton X-100 and 2 mMEDTA], (3) LiCl Wash Buffer [10 mM Tris-HCl (pH 8.0), 0.25 M LiCl, 0.5%NP-40, 0.5% sodium deoxycholate and 1 mM EDTA] and (4) 2 times with TEbuffer. The purified chromatin was eluted with 500 μl Elution Buffer (1%SDS and 0.1 M sodium bicarbonate at 65° C. for 15 min with gentleagitation in a gyratory shaking incubator. The eluted chromatin wasincubated with 0.2 M NaCl to reverse the protein-DNA cross-linking at65° C. overnight without agitation. Chromatin DNA was further purifiedby incubation with proteinase K (0.2 mg/mL) for 1 h to remove anyresidual proteins before the quantitative PCR analysis. Chromatinsamples without GFP antibody immunoprecipitation was used as control.C3H14 and MYB54 promoters were used as positive and negative control,respectively. Three biological replications were used in theexperiments.

Labeling for CBM3a and Immunofluorescence Microscopy Arabidopsisthaliana plants, ecotype Columbia (Col-0) wild type and 35S:AtMYB46transgenics, were grown on soil in a growth chamber (16 h light/8 hdark) at 23° C. for 8 weeks. Lower parts of the stems were fixed in FAAsolution (50% ethanol, 5% glacial acetic acid and 3.7% formaldehyde) for12 h at 4° C. After fixation, the fixed stems were embedded in paraffinand sectioned into 20 μm thin sections. The stem sections were labeledwith a crystalline cellulose-specific carbohydrate-binding module CBM3aas described by McCartney et al. (2004). In brief, the sections wereincubated in PBS containing 5% (w/v) milk protein (MP/PBS) and 10 μg/mlof the CBM3a for 1.5 h. Samples were then washed in PBS at least threetimes and incubated with a 100-fold dilution of mouse anti-hismonoclonal antibody (Sigma) in MP/PBS for 1.5 h. After washing with PBS,anti-mouse antibody linked to fluorescein isothiocyanate (anti-mouseFITC; Sigma) was applied for 1.5 h as a 50-fold dilution in MP/PBS indarkness. The samples were washed with PBS, mounted in a ProLong® Goldanti-fade solution (Invitrogen), and observed on a confocal laserscanning microscope, fitted with 488 nm laser and 505-550 nm band-pasfilter.Cell Wall Crystalline Cellulose Composition Analysis

Cell wall crystalline cellulose compositions were determined asdescribed previously (Ko et al., 2007). In brief, 3-weeks-old rosetteleaves were collected from soil grown wild-type. 35S:AtMYB46 andDEX-inducible MYB46 over-expression plants, and ground in liquidnitrogen using a mortar and pestle. The ground samples (60-70 mg) werewashed using 1.5 ml of 70% ethanol and centrifuged for 10 min at 10,000g. The pellets were washed with 1.5 ml of chloroform:methanol (1:1 v/v)and again with 500 μl acetone. The remaining pellet was considered to bethe cell walls and dried under nitrogen gas (N2). The cell wallmaterials were re-suspended in 250 μl of 2 M trifluoroacetic acid (TFA)and hydrolyzed for 90 min at 121° C. After the hydrolysis, samples werecentrifuged for 10 min at 10,000 g to separate a TFA-soluble fraction(non-cellulosic monosaccharides) and TFA-insoluble fraction (cellulose).The TFA-insoluble fraction was washed with 300 μl of 2-propanol andevaporated at 40° C. The washed samples were treated with Updegraffreagent (Acetic acid:nitric acid:water, 8:1:2 v/v/v) and heated inaluminum block for 30 min at 100° C. (Undegraff D M, 1969). Then, thesamples were centrifuged for 10 min at 10,000 g. The pellets were washedwith water once and then 3 times with acetone. Air-dried pellet wasSeaman hydrolyzed with 72% sulfuric acid for 30 min at room temperature(Selvendran and O'Neill, 1987). Final samples were precipitated for 5min at 10,000 g and analyzed with Anthrone method.

Example 2: MYB46 Expression Increases Cellulose Synthase Activity

This example shows that while many CESA genes exist in Arabidopsis, andwhile MYB46 may stimulate expression of a number of different types ofgenes, MYB46 stimulates expression of only three secondary wallcellulose synthases. The data provided herein demonstrates that suchup-regulation of these three CESA genes resulted in a substantialincrease (up to 30%) in crystalline cellulose content in transgenicArabidopsis plants.

Expression of the CESA4, CESA7 and CESA8 is up-regulated by MYB46

The inventors have identified a total of 37 genes whose expression maybe modulated by a master regulator of secondary wall formation, MYB46(At5g12870), in genome-wide survey of promoter sequences by using aMYB46-responsive cis-regulatory element (M46RE). Target genes that maybe modulated by MYB46 were selected based on three criteria: (1) theyhave at least one M46RE in the promoter region, (2) they areup-regulated by MYB46, and (3) they are co-expressed with MYB46.

Genes that can be regulated by MYB46 include all three secondary wallcellulose synthases, CESA4 (At5g4430), CESA7 (At5g17420) and CESA8(At4g18780). As a step toward verifying whether expression of CESA4(At5g4430), CESA7 (At5g17420) and CESA8 (At4g18780) can actually beregulated by MYB46, the inventors performed real time-PCR to examine theexpression pattern of these CESA genes in transgenic Arabidopsis plantsthat exhibited either constitutive or inducible over-expression ofMYB46, These real time-PCR analyses showed that all of the three CESAswere highly up-regulated by either constitutive or inducibleover-expression of MYB46 (FIG. 1).

Transcription of the CESA4, CESA7 and CESA8 is directly activated byMYB46

To investigate whether MYB46 can directly activate the transcription ofthese three CESAs, a steroid receptor-based inducible activation systemwas employed. In this system, a transcription factor fused with asteroid binding domain is sequestered in the cytoplasm by binding to acytoplasmic complex. Upon steroid treatment, the complex disrupts andthen transcription factor can enter the nucleus and regulate theexpression of downstream target genes. Coupled with a protein synthesisinhibitor, this steroid-mediated activation system has been widely usedto identify direct targets of a transcription factor in plants(Sablowski and Meyerowitz, 1998; Wagner et al., 1999; Baudry et al.,2004; Zhong et al., 2008).

In this study, MYB46 was fused with the regulatory region ofglucocorticoid receptor (MYB46-GR) and constitutively expressed as theMYB46-GR fusion protein under the control of CaMV 35S promoter inArabidopsis leaf protoplasts (FIG. 2A). As a positive control of theexperimental system, the promoter sequence of a known direct target,AtC3H14, of MYB46 (Ko et al., 2009) was used to drive a GUS reportergene. Upon dexamethasone (DEX) treatment, the MYB-GR chimeric proteinbecame functional to activate GUS reporter activity driven by theAtC3H14 promoter (FIGS. 2A, B). While the GUS activity induced by theDEX-activated MYB46-GR was completely abolished by cycloheximide (CHX)treatment, an inhibitor of protein synthesis (FIG. 2B), the expressionof the positive control AtC3H14 was clearly induced by the DEX-activatedMYB46-GR with CHX pretreatment (FIG. 2C). The induction level of AtC3H14by MYB46-GR was lower with the cycloheximide treatment compared to notreatment, which may reflect cycloheximide inhibition of the overallprotein synthesis, including that of MYB46-GR (FIG. 2C). Likewise, theDEX-activated MYB46-GR could activate the expression of all of the threesecondary wall CESA genes, even with the cycloheximide treatment (FIG.2D). This result indicates that MYB46 directly activates thetranscription of all of the three CESA genes tested.

MYB46 Binds to the Promoters of CESA4, CESA7 and CESA8 Genes

To confirm the physical interaction of MYB46 protein with the promoterregions of CESA4, CESA7 and CESA8 genes, we performed electrophoreticmobility shift assays (EMSA) using recombinant MYB46 proteins fused withglutathione S-transferase (GST-MYB46) and CESA promoter fragmentcontaining a M46RE motif (FIG. 3). Specific binding of MYB46 to the³²P-labeled promoter fragments, ProCESA4 (−248 to −69), ProCeA7 (−662 to−486), and ProCESA8 (−525 to −358) was established using; non-labeledpromoter fragments (e.g., ProCESA4_wt, FIG. 3A) as a competitor (FIG.3B). The binding specificity was further confirmed by using non-labeledpromoter fragments with single base mutation in the M46RE (e.g.,ProCESA4_m1 or m2) as a competitor. As expected, the MYB46 protein couldbind to the CESA promoter fragments while the GST protein alone couldnot bind to the fragments (FIG. 3B), demonstrating the interaction ofMYB46 protein with the promoters of the three CESA genes in vitro.

To further corroborate the interaction of MYB46 protein with the threeCESA promoters in vivo, the chromatin immunoprecipitation assay (ChIP)was performed using transgenic Arabidopsis plants that areover-expressing GFP-tagged MYB46 gene under the control of DEX-induciblepromoter (FIG. 4A). DEX treatment of the MYB46-GFP over-expressionplants caused ectopic secondary wall thickening in the leaf epidermaland mesophyll cells (data not shown), which is a typical phenotype ofectopic MYB46 over-expression as described previously (Ko et at, 2009).This indicates that the MYB46-GFP fusion protein can be used foranalysis of MYB46 binding sequences. Formaldehyde cross-linked chromatinfrom the leaf tissues collected from 3-week-old transgenic plants withor without DEX treatment was isolated and fragmented. Chromatinfragments from without DEX treatment were used as a negative control.MYB46-GFP-bound DNA fragments were immunoprecipitated by using GFPantibody and used as templates in the quantitative real-time PCRanalysis of CESA promoter sequences. All of the three CESA promoterswere highly enriched (3-8 fold) compared to control DNA (FIG. 4B). Inthe ChIP analysis, we used AtC3H14 and MYB54 as a positive and anegative control, respectively, since MYB54 is not a direct target ofMYB46.

Along with the finding that the expression of CESA4, CESA7 and CESA8 aredirectly activated by MYB46, these results provide both in vitro and invivo evidence that MYB46 directly binds to the promoter of all of thethree secondary wall-associated CESA genes to activate their expression.

Increase of Cellulose Contents by Up-Regulation of MYB46

MYB46 directly regulate the expression of CESA4, CESA7 and CESA8 genes.An increase of cellulose content may be observed when MYB46 expressionis increased. To test this hypothesis, the crystalline cellulose contentwas measured of transgenic Arabidopsis plants with either constitutiveor inducible over-expression of MYB46 (FIG. 5A). Compared to that ofwild-type plants, two independent lines of constitutive overexpressorsof MYB46 (OX8 and OX9) had a substantial increase (about 30%) increasein crystalline cellulose content in the leaf tissues of 3-week-oldplants. Furthermore, just 24-hr induction of MYB46 resulted in up to 27%increase compared to that of non-induced plants (FIG. 5A).

Crystalline cellulose accumulation in the stems of MYB46 overexpressorswas visualized by immune-histological staining of cellulose using CBM3a,a carbohydrate-binding module for crystalline cellulose (Blake et al.,2006). Compared to wild-type plants, fluorescent signal driven bycellulose accumulation was more evident in the xylem and interfascicularregions of the two constitutive MYB46 overexpressors (OX8 and OX9) (FIG.5B). Furthermore, in both of the two constitutive overexpressors,fluorescent signals were detected in epidermal cells where secondarywall formation does not occur normally, while no signals were noted inthe wild-type plants (FIG. 5B).

Taken together, these results confirm that ectopic up-regulation ofMYB46 resulted in substantial increase of cellulose contents throughactivation of the three secondary wall CESA genes in plants.

Example 3: HAM1 and HAM2 Transcription Factors Bind to CESA Promoters

This Example describes experiments illustrating that while the HAM1transcription factor binds to the CESA4 promoter, the HAM2 transcriptionfactor binds to both the CESA4 and the CESA7 promoters.

Procedures similar to those described in Examples 1 and 2 were used toascertain whether the HAM1 or HAM2 transcription factors physicallyinteract with any the promoter regions of CESA genes. Briefly,electrophoretic mobility shift assays (EMSA) were performed usingrecombinant HAM1 and HAM2 proteins fused with glutathione S-transferaseto ascertain whether these proteins hound to a selected CESA promoterfragment. Specific binding of HAM1 and HAM2 to the following ³²P-labeledpromoter fragments was tested: CESA4 Pro1 (−666 to −294), CESA4 Pro2(−248 to −1), and CesA7 Pro4 (−260 to −1). Binding was established usinga fifty-fold excess of corresponding non-labeled promoter fragment as acompetitor.

The HAM1 protein bound to the CESA4 Pro1 (−666 to −294) fragment but nosignificant binding was observed to the CESA4 Pro2 (−294 to −1) promoteror to the CesA7 Pro4 (−260 to −1) promoter fragment (FIG. 6).

In contrast, the HAM2 protein hound to the CESA4 Pro1 (−666 to −294) andProCeA7 (−260 to −1) promoter fragments, but no significant binding wasobserved to the CESA4 Pro2 (−294 to −1) promoter fragment (FIG. 6).

Example 4: MYB112 Transcription Factor Binds to a CESA Promoter

This Example describes experiments illustrating that the MYB112transcription factor binds to upstream regions of the CESA4 promoter.

Procedures similar to those described in Examples 1-3 were used toascertain whether the MY B112 transcription factor physically interactswith the promoter regions of CESA genes. Briefly, electrophoreticmobility shift assays (EMSA) were performed using recombinant MYB112protein fused with glutathione S-transferase to ascertain whether theMYB112 protein bound to a selected CESA promoter fragment. Specificbinding of MYB112 to the following ³²P-labeled promoter fragments wastested: CESA4 Pro1 (−666 to −294) and CESA4 Pro2 (−294 to −1). Bindingwas established using a ten- or fifty-fold excess of correspondingnon-labeled promoter fragment as a competitor.

The MYB112 protein bound to the CESA4 Pro1 (−666 to −294) fragment butno significant binding was observed to the CESA4 Pro2 (−294 to −1)promoter fragment (FIG. 7).

Example 5: The WRKY11 Transcription Factor Binds to a CESA Promoter

This Example describes experiments illustrating that the WRKY11transcription factor binds to upstream regions of the CESA4 promoter.

Procedures similar to those described in Examples 1-4 were used toascertain whether the WRKY11 transcription factor physically interactswith the promoter regions of CESA genes. Briefly, electrophoreticmobility shift assays (EMSA) were performed using recombinant WRKY11protein fused with glutathione S-transferase to ascertain whether theWRKY11 protein bound to a selected CESA promoter fragment. Specificbinding of MYB112 to the following ³²P-labeled promoter fragments wastested: CESA4 Pro1 (−666 to −294) and CESA4 Pro2 (−294 to −1). Bindingwas established using a fifty-fold excess of corresponding non-labeledpromoter fragment as a competitor.

The WRKY11 protein bound to the CESA4 Pro1 (−666 to −294) fragment butno significant binding was observed to the CESA4 Pro2 (−294 to −1)promoter fragment (FIG. 8).

Example 6: The ERF6 Transcription Factor Binds to a CESA Promoter

This Example describes experiments illustrating that the ERF6transcription factor binds to upstream regions of the CESA4 promoter.

Procedures similar to those described in Examples 1-5 were used toascertain whether the ERF6 transcription factor physically interactswith the promoter regions of CESA genes. Briefly, electrophoreticmobility shift assays (EMSA) were performed using recombinant ERF6protein fused with glutathione S-transferase to ascertain whether theERF6 protein bound to a selected CESA promoter fragment. Specificbinding of ERF6 to the following ³²P-labeled promoter fragments wastested: CESA4 Pro1 (−666 to −294) and CESA4 Pro2 (−294 to −1). Bindingwas established using a fifty-fold excess of corresponding non-labeledpromoter fragment as a competitor.

The ERF6 protein bound to the CESA4 Pro1 (−666 to −294) fragment but nosignificant binding was observed to the CESA4 Pro2 (−294 to −1) promoterfragment (FIG. 9).

Example 7: MYB46 is Needed for Expression of Secondary Wall-AssociatedCellulose Synthases in Arabidopsis

This Example further illustrates the function of MYB46 and demonstratesthat it is a key transcription factor for up-regulation of CESA4, CESA7and CESA8 gene expression.

Materials and Methods

Plant Materials and Growth Conditions.

Arabidopsis thaliana ecotype Columbia (Col-0) and three T-DNAinsertional mutants of cesa [cesa4 (SALK_084627), cesa7 (SALK_029940)and cesa8 (SALK_026812)](FIG. 10) were used in the experiments. Plantswere grown on soil in a growth chamber (16 h light/8 h dark) at 23° C.All experiments were performed in triplicates and repeated at leastthree times.

Plasmids Construction and Plant Transformation.

All of the constructs used in this study were verified by DNAsequencing. The coding regions of CESA4 (At5g44030), CESA7 (At5g17420)and CESA8 (At4g18780) were obtained by PCR amplification from steincDNAs of Arabidopsis. For the genetic complementation, the PCR-amplifiedcoding region was fused with either native or mutated promoter from theCESA genes (FIG. 11). The mutated promoter was created by PCR-basedpoint mutations of the two base pairs critical in the M46RE (Kim et al.,Plant Molecular Biology 78: 489-501 (2012)) as shown in FIG. 11. Theprimers used in this experiment are listed in Table 1.

TABLE 1 Primer sequences SEQ Primers Sequence ID NO:CESA4 - AGI No. AT5G44030 Pro-ForwardCCCACTAGTTAAATCTTATTTACTAACAAAACAATAAGA 22 Pro-ReverseCCCCTCGAGGGCGAGGTACACTGAGCTC 23 Pro-Mutation-F1 GATTCAAGAACATAGCCAGATTTTT TAAAGT 24 Pro-Mutation-R1 TCTTACTTAATATTTTGTATCTTATAAACTTTAAAAAATCT25 Pro-Mutation-F2 TGAGCTGTCTCCTTCTTCCA AA AAATCT 26 Pro-Mutation-R2TTCAAGAGACAGCAACAAGATTT TT TGGAAG 27 Pro-Mutation-F3GACCCAATTTCACTCACAGTTTTTTACAAC 28 Pro-Mutation-R3GTTGTGAAGAAAACTGAGGTTGTAAAAAACTG 29 CDS-F1 CCCCTCGAGATGGAACCAAACACCATGG30 CDS-R1 GTACTGCAGAGACTCGAACCA 31 CDS-F2 TCTCTGCAGTACTCACTAATGCTC 32CDS-R2 CCCACTAGTTTAACAGTCGACGCCACAT 33 RT-ForwardCAACAGATGATGATGACTTTGGA 34 RT-Reverse AGACCTTTGAGGAATGGGTAGAG 35 SALK RGGACGCCATTGCTGCTTACTGTTG 79 CESA7 - AGI No. AT5G17420 Pro-ForwardCCCGAGCTCAGATTGAGGATCATTTTATTTATTTATTAG 36 Pro-ReverseCCCCTCGAGAGGGACGGCCGGAGA 37 Pro-Mutation-F1 TAGCTTATGTATGCAGAAAATTCA AATAATTA 38 Pro-Mutation-R1 GTTACGTTCCCTGTCCTTAATTATTTGAATT 39Pro-Mutation-F2 TGGCTTGCACTCCTCTCA AA AAACCT 40 Pro-Mutation-R2AAATTAGTTAGGGGGTAAGGTTTTTTGAGAG 41 CDS-F1 CCCCTCGAGATGGAAGCTAGCGCCG 42CDS-R1 TGAGGATCCATCAAAAAACAC 43 CDS-F2 GATGGATCCTCAGATTGGAA 44 CDS-R2CCCGAGCTCTCAGCAGTTGATGCCACA 45 RT-Forward CAACAGATGATGATGACTTTGGA 46RT-Reverse AGACCTTTGAGGAATGGGTAGAG 47 SALK R GCAAGCTACGAAGAGGTCTCC 48CESA8 - AGI No. AT4G18780 Pro-Forward CCCACTAGTTGATGGATGGTTTTGCTGTA 49(TAA) Pro-Reverse (TAA) CCCCTGCAGCTTCGAATTCCCCTGTTTG 50 Pro-Mutation-F1GATTTTAATTCTTATTTTTCTTATAGAAAGTT TT TGATTG 51 Pro-Mutation-R1TTATAATTTTTAAGTAAATCTTTTCAATCAAAAACTTT 52 Pro-Mutation-F2TCCGATTTTTCACAATCCA AA AAACTT 53 Pro-Mutation-R2AGGAAAAAAAGTTATTAAAAAAAGTTTTTTGGATT 54 CDS-F1CCCCTGCAGATGATGGAGTCTAGGTCTCCC 55 CDS-R1 ACAGGATCCATTAAAAAGCAC 56 CDS-F2AATGGATCCTGTTGTTGGTC 57 CDS-R2 CCCACTAGTTTAGCAATCGATCAAAAGACAG 58RT-Forward CGATGTTAATATGAGAGGGCTTG 59 RT-Reverse GGAAGGATCTTGAGGTTGTTTCT60 SALK R GTACTTATATGTCTAGCATGAATCCCTG 61 Left-border primerATTTTGCCGATTTCGGAAC 62 ACT8 - ACI No. AT1G49240 RT-ForwardATGAAGATTAAGGTCGTGGCA 63 RT-Reverse TCCGAGTTGAAGAGGCTAC 64 CDS, codingsequence; Pro, promoter; Underlined letters indicate the restrictionenzyme sites used for the cloning into the vector; Underlined and boldletters indicate the point mutations introduced.The resulting promoter-CESA construct was introduced into a binaryvector pCB308 (Xiang et al., Plant Molecular Biology 40: 711-717 (1999))and used in the Agrobacterium-mediated transformation of both wild-typeArabidopsis (Col-0) plants and cesa T-DNA insertion mutants.Homozygocity of these cesa mutants and their genetic complementationwere confirmed by polymerase chain reaction amplification of the genomicDNA (FIG. 12).

RNA extraction and RT-PCR.

Total RNAs were extracted using Plant RNeasy extraction kit (Qiagen)according to the manufacturer's protocol. For RT-PCR analysis, totalRNAs were first treated with DNaseI before the first-strand cDNAsynthesis by SuperScript II Reverse Transcriptase (Invitrogen). RT-PCRwas carried out using 1 μL of the reaction products as a template.Amplified DNA fragments were separated on 1% agarose gel and stainedwith ethidium bromide. The primers used for RT-PCR are shown in Table 1.

Histological Analysis.

The stem area located immediately above the rosette leaves (basal level)was cross-sectioned using Microtome (Leica RM2025) into thin sections (5μm thick) and paraffin embedded as described previously (Ko et al., 2004and 2007). The sections were then stained with 0.05% toluidine blue Ofor 1 min to visualize secondary xylem.

Results

T-DNA insertional mutants of three secondary wall cesa (cesa4, cesa7,and cesa8) were obtained from Arabidopsis Biological Resource Center(see website at abrc.osu.edu/) (FIG. 10). All of the mutants displayedphenotypes such as collapsed/irregular xylem and pendent stem (FIG. 13and FIG. 14). The three CESAs (CESA4, CESA7 and CESA8) are required forcellulose synthesis in the secondary walls of Arabidopsis plants. Eachof these three CESA genes appears to be equally important in thefunction of the cellulose synthase complex and one cannot substitute foranother (Gardiner et al., Plant Cell 15: 1740-1748 (2003)). Therefore,even a single T-DNA insertion mutation of one the three CESA genesresults in a severe phenotype (FIGS. 13 and 14).

Cells from each cesa mutant plant type were transformed with thecorresponding CESA wild type coding region operably linked to either itsnative promoter or a mutated promoter. The mutated promoters had pointmutations in the cis-regulatory element, M46RE, which is recognized byMYB 46, as shown in Table 2.

TABLE 2 Summary of Promoter Sequences Promoter Type Wild Type MutantCES44 (−404 to −397) ATTTGGTA ATTTTTTA SEQ ID NO: 65 SEQ ID NO: 72CESA4 (−218 to −211) CACCAAAT CAAAAAAT SEQ ID NO: 66 SEQ ID NO: 73CESA4 (−150 to −143) GTTTGGTA GTTTTTTA SEQ ID NO: 67 SEQ ID NO: 74CESA7 (−597 to −590) CACCTAAT CAAATAAT SEQ ID NO: 68 SEQ ID NO: 75CESA7 (−553 to −546) CACCAAAC CAAAAAAC SEQ ID NO: 69 SEQ ID NO: 76CESA8 (−446 to −439) AGTTGGTG AGTTGGTG SEQ ID NO: 70 SEQ ID NO: 77CESA8 (−140 to −133) CACCAAAC CAAAAAAC SEQ ID NO: 71 SEQ ID NO: 78These mutations effectively eliminated MYB46 binding (Kim et al., PlantMolecular Biology 78: 489-501 (2012), herein incorporated by referencein its entirety), and resulted in failure of CESA expression (FIG. 13).

Both the wild-type and vector control plants grew upright and werenormal in appearance. In contrast, the cesa mutants exhibited retardedgrowth and the characteristic ‘pendent stem’ phenotype (FIG. 13), withcollapsed xylem (FIG. 14). Transgenic plants expressing nativepromoter-driven CESAs restored wild-type phenotype. However, geneticcomplementation with the mutant promoters that were not recognized byMYB46 exhibited the mutant phenotype (i.e., pendent stem and collapsedxylem phenotype) (FIGS. 13 and 14). These results indicate that MYB46binding to the M46RE site is required for functional expression of thesecondary wall CESAs in planta.

Transcription factor MYB46 and its orthologs have been shown to bemaster switches for the biosynthesis of the three major components ofsecondary walls (e.g., cellulose, hemicellulose, and lignin) inArabidopsis, poplar, rice and maize. Furthermore, MYB46 has recentlybeen shown to be a direct regulator of all three secondary wall CESAgenes (CESA4, CESA7 and CESA8) (Kim et al., Plant J 73: 26-36 (2013),herein incorporated by reference in its entirety). Transcription factorMYB83 (NM_111685.2; GI:145338258), a homolog of MYB46, is functionallyredundant with MYB46 and also operates by binding to M46RE. Doubleknockout of myb46/myb83 does not produce any viable plants (unpublishedobservation). In light of these observations, MYB46 plays a key role inthe biosynthesis of secondary wall cellulose biosynthesis. However, thefinding that MYB46/MYB83 is required for functional expression of allthree secondary wall CESA genes is significant. Considering theimportance of secondary wall cellulose synthesis for the growth andsurvival of the plant, additional regulators may operate in concert withMYB46 and/or may be involved in the transcriptional regulation ofsecondary wall CESA genes. In fact, the inventors have recently reportedseveral candidate regulators (e.g., MYB112, WRKY11 and ERF6)) of CESA4,albeit none of them appears to be involved in the MYB46-mediatedregulation pathway (Kim et al., 2013). Some of the secondary wall NACtranscription factors such as VND6, VND7, NST1 and NST2 bind to animperfect palindromic 19-bp consensus sequence (SNBE), which is similarto M46RE (Zhong et al., 2010). Recently, Ohashi-Ito et al. (2010)reported the binding of VND6 to the promoter of CESA4. VND7 was alsosuggested as a direct regulator of CESA4 and CESA8 (Yamaguchi et al.,2011). However, none of the secondary wall CESA genes was directlyinduced by estradiol-activated VND7 (Zhong et al., 2010). The presenceof multiple regulators supports the notion that the transcriptionalregulation of cellulose biosynthesis is multifaceted and complex.

So far, MYB46/MYB83 is the only transcription factor shown to be directregulator of all three secondary wall CESAs. The fact that MYB46 isrequired for functional expression of the three secondary wall CESAsindicates that MYB46 is necessary component of the transcriptionalregulatory complex for the CESA regulation.

REFERENCES

-   1. Delmer D P (1999) CELLULOSE BIOSYNTHESIS: Exciting Times for A    Difficult Field of Study. Annu Rev Plant Physiol Plant Mol Biol 50:    245-276.-   2. Ragauskas A J, et al. (2006) The path forward for biofuels and    biomaterials. Science 311 (5760): 484-489.-   3. Pear J R, Kawagoe Y, Schreckengost W E, Delmer D P, & Stalker D    M (1996) Higher plants contain homologs of the bacterial celA genes    encoding the catalytic subunit of cellulose synthase. Proc Natl Acad    Sci USA 93 (22): 12637-12642.-   4. Somerville C (2006) Cellulose synthesis in higher plants. Anna    Rev Cell Dev Biol 22: 53-78.-   5. Endler A & Persson S (2011) Cellulose synthases and synthesis in    Arabidopsis. Mol Plant 4 (2): 199-211.-   6. Richmond T A & Somerville C R (2001) Integrative approaches to    determining Cs1 function. Plant Mol Biol 47(1-2): 131-143.-   7. Doblin M S, Kurek I, Jacob-Wilk D, & Delmer D P (2002) Cellulose    biosynthesis in plants: from genes to rosettes. Plant Cell Physiol    43(12): 1407-1420.-   8. Williamson R E, Burn J E, & Hocart C H (2002) Towards the    mechanism of cellulose synthesis. Trends Plant Sci 7(10): 461-467.-   9. Kumar M, et al. (2009) An update on the nomenclature for the    cellulose synthase genes in Populus. Trends Plant Sci 14(5):    248-254.-   10. Sjöström E (1992). Wood Chemistry Fundamentals and Applications,    second edition. Academic Press. San Diego.-   11. Carpita N & McCann M (2000) The cell wall. In Biochemistry and    Molecular Biology of Plants. Edited by Buchanan B B, Gruissem W,    Jones R L pp. 52-108. American Society of Plant Physiologists,    Rockville, Md.-   12. Somerville C, et al. (2004) Toward a systems approach to    understanding plant cell walls. Science 306(5705): 2206-2211.-   13. Cosgrove D J (2005) Growth of the plant cell wall. Nat Rev Mol    Cell Biol 6(11): 850-861.-   14. Demura T & Ye Z H (2010) Regulation of plant biomass production.    Curr Opin Plant Biol 13(3): 299-304.-   15. Ko J H, Yang S H, Park A H, Lerouxel O, Han K H (2007) ANAC012,    a member of the plant-specific NAC transcription factor family,    negatively regulates xylary fiber development in Arabidopsis    thaliana. Plant J 50(6): 1035-1048.-   16. Ko J H, Kim W C, & Han K H (2009) Ectopic expression of MYB46    identifies transcriptional regulatory genes involved in secondary    wall biosynthesis in Arabidopsis. Plant J 60(4): 649-665.-   17. Mitsuda N, Seki M, Shinozaki K, & Ohme-Takagi M (2005) The NAC    transcription factors NST1 and NST2 of Arabidopsis regulate    secondary wall thickenings and are required for anther dehiscence.    Plant Cell 17(11): 2993-3006.-   18. Mitsuda N, et al. (2007) NAC transcription factors, NST1 and    NST3, are key regulators of the formation of secondary walls in    woody tissues of Arabidopsis. Plant Cell 19(1): 270-280.-   19. Zhong R & Ye Z H (2007) Regulation of cell wall biosynthesis.    Curr Opin Plant Biol 10(6): 564-572.-   20. Zhong R, Richardson E A, & Ye Z H (2007) The MYB46 transcription    factor is a direct target of SND1 and regulates secondary wall    biosynthesis in Arabidopsis. Plant Cell 19(9): 2776-2792.-   21. Zhong R, Lee C, Zhou J, McCarthy R L, & Ye Z H (2008) A battery    of transcription factors involved in the regulation of secondary    cell wall biosynthesis in Arabidopsis. Plant Cell 20(10): 2763-2782.-   22. Zhong R, Lee C, & Ye Z H (2010) Evolutionary conservation of the    transcriptional network regulating secondary cell wall biosynthesis.    Trends Plant Sci 15(11): 625-632.-   23. Taylor N G, Howells R M, Huttly A K, Vickers K, & Turner S    R (2003) Interactions among three distinct CesA proteins essential    for cellulose synthesis. Proc Natl Acad Sci USA 100(3): 1450-1455.-   24. Sablowski R W &. Meyerowitz E M (1998) A homolog of NO APICAL    MERISTEM is an immediate target of the floral homeotic genes    APETALA3/PISTILLATA. Cell 92(1): 93-103.-   25. Wagner D, Sablowski R W, & Meyerowitz E M (1999) Transcriptional    activation of APETALA1 by LEAFY. Science 285(5427): 582-584.-   26. Baudry A, et al. (2004) TT2, TT8, and TTG1 synergistically    specify the expression of BANYULS and proanthocyanidin biosynthesis    in Arabidopsis thaliana. Plant J 39(3):366-380.-   27. Blake A W, et al. (2006) Understanding the biological rationale    for the diversity of cellulose-directed carbohydrate-binding modules    in prokaryotic enzymes. J Biol Chem 281(39): 29321-29329.-   28. Joshi C P, et al. (2004) Genomics of cellulose biosynthesis in    poplars. New Phytol 164:53-61.-   29. Joshi C P & Mansfield S D (2007) The cellulose paradox—simple    molecule, complex biosynthesis. Curr Opin. Plant Biol 10(3):    220-226.-   30. McCarthy R L, et al. (2010) The poplar MYB transcription    factors, PtrMYB3 and PtrMYB20, are involved in the regulation of    secondary wall biosynthesis. Plant Cell Physiol 51(6): 1084-1090.-   31. Zhong R, et al. (2011) Transcriptional activation of secondary    wall biosynthesis by rice and maize NAC and MYB transcription    factors. Plant Cell Physiol 52(10): 1856-1871.-   32. Zhou J, Lee C, Zhong R, & Ye Z H (2009) MYB58 and MYB63 are    transcriptional activators of the lignin biosynthetic pathway during    secondary cell wall formation in Arabidopsis. Plant Cell    21(1):248-266.-   33. McCarthy R L, Zhong R, & Ye Z H (2009) MYB83 is a direct target    of SND1 and acts redundantly with MYB46 in the regulation of    secondary cell wall biosynthesis in Arabidopsis. Plant Cell Physiol    50(11): 1950-1964.-   34. Zhong R, Ye Z-H. (2012) MYB46 and MYB83 bind to the SMRE sites    and directly activate a suite of transcription factors and secondary    wall biosynthetic genes. Plant and Cell Physiology 53: 368-380.-   35. Zhong R, Morrison W H, III, Freshour G D, Hahn M G, Ye    Z-H. (2003) Expression of a mutant form of cellulose synthase    AtCesA7 causes dominant negative effect on cellulose biosynthesis.    Plant Physiology 132: 786-795.-   36. Yamaguchi M, Mitsuda N, Ohtani M, Ohme-Takagi M, Kato K,    Demura T. (2011) VASCULAR-RELATED NAC-DOMAIN7 directly regulates the    expression of a broad range of genes for xylem vessel formation. The    Plant Journal 66: 579-590.-   37. Xiang C, Han P, Lutziger I, Oliver D J. (1999) A mini binary    vector series for plant transformation. Plant Molecular Biology 40:    711-717.-   38. Turner S R, Somerville C R. (1997) Collapsed xylem phenotype of    Arabidopsis identifies mutants deficient in cellulose deposition in    the secondary cell wall. Plant Cell 9: 689-701.-   39. Taylor N, Gardiner J, Whiteman R, Turner S. (2004) Cellulose    synthesis in the Arabidopsis secondary cell wall. Cellulose 11:    329-338.-   40. Taylor N G, Scheible W R, Cutler S, Somerville C R, Turner    S R. (1999) The irregular xylem3 locus of Arabidopsis encodes a    cellulose synthase required for secondary cell wall synthesis. Plant    Cell 769-780.-   41. Ohashi-Ito K, Oda Y. Fukuda H. (2010) Arabidopsis    VASCULAR-RELATED NAC-DOMAIN6 directly regulates the genes that    govern programmed cell death and secondary wall formation during    xylem differentiation. Plant Cell 22: 3461-3473.-   42. Ko J-H, Kim W-C, Kim J-Y, Ahan S J, Han K-H. (2012)    MYB46-mediated transcriptional regulation of secondary wall    biosynthesis. Molecular Plant 5: 961-962.-   43. Ko J-H, Han K.-H, Park S, Yang J. (2004) Plant body    weight-induced secondary growth in Arabidopsis and its transcription    phenotype revealed by whole-transcriptome profiling. Plant    Physiology 135: 1069-1083.-   44. Kim W-C, Ko J-H, Kim J-Y, Kim J M, Bae H J, Han K-H. (2013)    MYB46 directly regulates the gene expression of secondary    wall-associated cellulose synthases in Arabidopsis. Plant J 73:    26-36.-   45. Kim W-C, Ko J-H, Han K. H. (2012) Identification of a cis-acting    regulatory motif recognized by MYB46, a master transcriptional    regulator of secondary wall biosynthesis. Plant Molecular Biology    78: 489-501.-   46. Gardiner J C, Taylor N G, Turner. S R. 2003. Control of    cellulose synthase complex localization in developing xylem. Plant    Cell 15: 1740-1748.-   47. Brown R M. (2004) Cellulose structure and biosynthesis: what is    in store for the 21st century? Journal of Polymer Science. Part A.    Polymer Chemistry 42: 487-495.

All patents and publications referenced or mentioned herein areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each such referenced patent or publicationis hereby specifically incorporated by reference to the same extent asif it had been incorporated by reference in its entirety individually orset forth herein in its entirety. Applicants reserve the right tophysically incorporate into this specification any and all materials andinformation from any such cited patents or publications.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and the methods and processes are not necessarilyrestricted to the orders of steps indicated herein or in the claims. Asused herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, a reference to “a nucleic acid” or “apolypeptide” includes a plurality of such nucleic acids or polypeptides(for example, a solution of nucleic acids or polypeptides or a series ofnucleic acid or polypeptide preparations), and so forth. Under nocircumstances may the patent be interpreted to be limited to thespecific examples or embodiments or methods specifically disclosedherein. Under no circumstances may the patent be interpreted to belimited by any statement made by any Examiner or any other official oremployee of the Patent and Trademark Office unless such statement isspecifically and without qualification or reservation expressly adoptedin a responsive writing by Applicants.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims and statements of theinvention.

The following statements of the invention are intended to describe andsummarize various embodiments of the invention according to theforegoing description in the specification.

Statements Describing Aspects of the Invention

-   -   1. A method of increasing expression of a cellulose synthase        gene in a plant comprising providing conditions in the plant for        a transcription factor to bind to a promoter or enhancer region        operably linked to a coding region of the cellulose synthase,        wherein the transcription factor is selected from the group        consisting of MYB46, HAM1, HAM2, MYB112, WRKY11, ERF6, and any        combination thereof.    -   2. The method of statement 1, wherein providing conditions for a        transcription factor to bind to a promoter or enhancer comprises        transforming cells of the plant with a transgene encoding the        transcription factor and/or generating a plant from plant cells        comprising the isolated nucleic encoding the transcription        factor.    -   3. The method of statement 1 or 2, wherein providing conditions        for a transcription factor to bind to a promoter or enhancer        comprises transforming cells of the plant with a transgene        encoding the transcription factor wherein the transgene        comprises a transgene promoter segment operably linked to a        nucleic acid segment encoding the transcription factor.    -   4. The method of statement 3, wherein the transgene promoter        segment is heterologous to the transcription factor's native        gene.    -   5. The method of statement 3 or 4, wherein the transgene        promoter segment is a strong promoter, weak promoter, inducible        promoter, tissue specific promoter, developmentally regulated        promoter or a combination thereof.    -   6. The method of any of statements 1-5, wherein the promoter or        enhancer region operably linked to the cellulose synthase gene        is the gene's native promoter.    -   7. The method of any of statements 1-6 wherein the promoter or        enhancer region operably linked to the cellulose synthase gene        is a nucleic acid segment with a sequence comprising any of SEQ        ID NOs: 3-11, 65-71, or any combination thereof.    -   8. The method of any of any of statements 1-7, wherein the        transcription factor has an amino acid sequence with at least        75% sequence identity, or at least 80% sequence identity, or at        least 90% sequence identity, or at least 95% sequence identity        to an amino acid sequence comprising any of SEQ ID NOs: 2, 13,        15, 17, 19, 21 or a combination thereof.    -   9. The method of any of any of statements 1-8, wherein the        transcription factor has an amino acid sequence comprising or        consisting essentially of any of SEQ ID NOs: 2, 13, 15, 17, 19,        21 or any combination thereof.    -   10. The method of any of statements 1-9, wherein the cellulose        synthase is active in synthesizing secondary wall cellulose.    -   11. The method of any of statements 1-10, wherein the cellulose        synthase is a CESA4.    -   12. The method of any of statements 1-11, wherein the cellulose        synthase is a CESA4 gene with a promoter having a nucleotide        sequence selected from the group consisting of any of SEQ ID        NO:3-5, 65-67, and any combination thereof.    -   13. The method of any of statements 1-12, wherein the cellulose        synthase is a CESA4 and the CESA4 expression is increased by a        transcription factor selected from the group consisting of        MYB46, HAM1, HAM2, MYB112, WRKY11, ERF6 and any combination        thereof.    -   14. The method of any of statements 1-13, wherein the cellulose        synthase is a CESA7.    -   15. The method of any of statements 1-14, wherein the cellulose        synthase is a CESA7 gene with a promoter having a nucleotide        sequence selected from the group consisting of any of SEQ ID        NO:6-8, 68, 69, and any combination thereof.    -   16. The method of any of statement 1-5, wherein the cellulose        synthase is a CESA7 and the CESA7 expression is increased by a        transcription factor selected from the group consisting of        MYB46, HAM2 and a combination thereof.    -   17. The method of any of statements 1-16, wherein the cellulose        synthase is a CESA8.    -   18. The method of any of statements 1-17, wherein the cellulose        synthase is a CESA8 gene with a promoter having a nucleotide        sequence selected from the group consisting of any of SEQ ID        NO:9-11, 70, 71, and any combination thereof.    -   19. The method of any of statements 1-18, wherein the cellulose        synthase is a CESA8 and the CESA8 expression is increased by a        MYB46 transcription factor.    -   20. An isolated nucleic acid encoding a plant gene promoter or a        plant gene enhancer comprising a nucleotide sequence selected        from the group consisting of any of SEQ ID NOs: 3-11, 65-71, or        a combination thereof.    -   21. A transgene comprising a plant gene promoter or a plant gene        enhancer comprising a nucleotide sequence selected from the        group consisting of any of SEQ ID NOs: 3-11, 65-71, or a        combination thereof.    -   22. A transgene comprising a transgene promoter segment and a        segment encoding a plant transcription factor selected from the        group consisting of MYB46, HAM1, HAM2, MBY112, WRKY11, ERF6, or        a combination thereof.    -   23. The transgene of statement 22, wherein the transgene        promoter segment is heterologous to the transcription factor's        native gene.    -   24. The transgene of statement 22 or 23, wherein the transgene        promoter segment is a strong promoter, weak promoter, inducible        promoter, tissue specific promoter, developmentally regulated        promoter or a combination thereof.    -   25. The transgene of any of statements 22-24, wherein the        transcription factor has an amino acid sequence comprising any        of SEQ ID NOs: 2, 13, 15, 17, 19, 21 or a combination thereof.    -   26. A kit comprising:        -   a. a container comprising an isolated nucleic acid encoding            a plant gene promoter or a plant gene enhancer comprising a            nucleotide sequence selected from the group consisting of            any of SEQ ID NOs: 3-11, 65-71, or a combination thereof;            and        -   b. instructions for operably linking the isolated promoter            or enhancer nucleic acid to a selected coding region.    -   27. The kit of statement 26, wherein the instructions comprise a        method for operably linking the isolated promoter or enhancer        nucleic acid to a selected coding region in vitro.    -   28. The kit of statement 26, wherein the instructions comprise a        method for operably linking the isolated promoter or enhancer        nucleic acid to a selected coding region in vivo.    -   29. The kit of any of statements 26-28, wherein the selected        coding region is a plant gene coding region.    -   30. The kit of any of statements 26-29, wherein the selected        coding region is a plant cellulose synthase gene coding region.    -   31. The kit of any of statements 26-30, further comprising a        second container comprising an isolated nucleic acid encoding is        a plant cellulose synthase.    -   32. The kit of any of statements 26-31, wherein the isolated        nucleic acid encoding is a plant cellulose synthase in the        second container comprises a heterologous promoter segment and a        segment encoding the plant cellulose synthase.    -   33. The kit of statement 32, wherein the heterologous promoter        is a strong promoter, weak promoter, inducible promoter, tissue        specific promoter, developmentally regulated promoter or a        combination thereof.    -   34. A plant comprising an isolated nucleic acid encoding a plant        transcription factor selected from the group consisting of        MYB46, HAM1, HAM2, MYB112, WRKY11, ERF6, or a combination        thereof.    -   35. The plant of statement 34, wherein the isolated nucleic acid        comprises a heterologous promoter segment operably linked to a        nucleic segment that encodes the plant transcription factor        coding region.    -   36. The plant of statement 34 or 35, wherein the heterologous        promoter is not the plant transcription factor's natural        promoter.    -   37 The plant of statement 36, wherein the heterologous promoter        is a strong, weak, inducible, tissue specific, developmentally        regulated or a combination thereof.    -   38. The plant of any of statements 34-37, wherein the isolated        nucleic acid expresses increased levels of the plant        transcription factor in the plant compared to a corresponding        transcription factor gene naturally present in a wild type plant        of the same species.    -   39. The plant of any of statements 34-38, wherein the plant has        increased levels of secondary wall cellulose compared to a wild        type plant of the same species without the isolated nucleic        acid.    -   40. The plant of any of statements 34-39, wherein the plant has        at least about 1%, at least about 2%, at least about 3%, at        least about 4%, at least about 5%, at least about 10%, at least        about 15%, at least about 20%, at least about 25%, at least        about 30% increased cellulose content compared to a wild type        plant of the same species that does not have the isolated        nucleic acid.    -   41. The plant of any of statements 34-40, wherein the plant is a        transgenic plant, a genetically modified plant, or a plant        selectively bred to comprise the isolated nucleic acid.    -   42. The plant of any of statements 34-41, wherein the plant        transcription factor is MYB46.    -   43. The plant of any of statements 34-42, wherein the plant is a        grass species, softwood species, or hardwood species.    -   44. The plant of any of statements 34-43, wherein the plant        grass species is maize, barley, oats, rice, sorghum, millet,        rye, switchgrass, prairie grass, wheat grass, sudangrass,        sorghum, and straw-producing plants.    -   45. The plant of any of statements 34-44, wherein the plant is a        poplar species, pine species, or eucalyptus species.    -   46. A seed comprising an isolated nucleic acid encoding a plant        transcription factor selected from the group consisting of        MYB46, HAM1, HAM2, MYB112, WRKY11, ERF6, or a combination        thereof.    -   47. The seed of statement 46, wherein the isolated nucleic acid        comprises a heterologous promoter segment operably linked to a        nucleic segment that encodes the plant transcription factor        coding region.    -   48. The seed of statement 47, wherein the heterologous promoter        is not the plant transcription factor's natural promoter.    -   49. The seed of statement 47 or 48, wherein the heterologous        promoter is a strong, weak, inducible, tissue specific,        developmentally regulated or a combination thereof.    -   50. The seed of any statements 46-49, wherein the plant is a        grass species, softwood species, or hardwood species.    -   51. The seed of any statements 46-50, wherein the plant grass        species is maize, barley, oats, rice, sorghum, millet, rye,        switchgrass, prairie grass, wheat grass, sudangrass, sorghum,        and straw-producing plants.    -   52. The seed of any statements 46-51, wherein the plant is a        poplar species, pine species, or eucalyptus species.    -   53. A plant biomass comprising secondary wall cellulose isolated        from a plant comprising an isolated nucleic acid encoding a        plant transcription factor selected from the group consisting of        MYB46, HAM1, HAM2, MYB112, WRKY11, ERF6, or a combination        thereof.    -   54. A kit comprising:        -   a. a container comprising an isolated nucleic acid encoding            a plant gene promoter or a plant gene enhancer comprising a            nucleotide sequence selected from the group consisting of            any of SEQ ID NOs: 3-11, 65-71, or a combination thereof,            operably linked to an isolated nucleic acid comprising a            coding region of a plant cellulose synthase, and        -   b. instructions for transforming a plant cell with the            isolated nucleic acid to generate a transformed plant cell.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings faking within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group. Other embodiments are described within thefollowing claims.

What is claimed:
 1. A plant comprising a heterologous nucleic acidcomprising a tissue specific promoter operably linked to a nucleic acidsegment encoding a WRKY11 plant transcription factor having at least 95%sequence identity to SEQ ID NO:19, and wherein the plant has at least 4%increased levels of secondary wall cellulose compared to a wild typeplant of the same species without the heterologous nucleic acid.
 2. Theplant of claim 1, wherein the plant is a softwood plant or hardwoodplant.
 3. The plant of claim 1, wherein the plant is a poplar species,pine species, or eucalyptus species plant.
 4. The plant of claim 1,wherein the promoter is a xylem-specific promoter.
 5. The plant of claim1, wherein the heterologous nucleic acid expresses increased levels ofthe WRKY11 plant transcription factor in the plant compared to acorresponding transcription factor gene naturally present in a wild typeplant of the same species.
 6. The plant of claim 1, wherein the plant isa transgenic plant, a genetically modified plant, or a plant selectivelybred to comprise the heterologous nucleic acid.
 7. A seed from the plantof claim
 1. 8. A plant biomass comprising secondary wall celluloseisolated from a plant comprising a heterologous nucleic acid comprisinga tissue specific promoter segment operably linked to a nucleic acidsegment encoding a WRKY11 plant transcription factor, wherein the WRKY11has at least 95% sequence identity to SEQ ID NO:19.
 9. A method ofincreasing cellulose content in a plant comprising expressing WRKY11 inthe plant from a heterologous nucleic acid comprising a heterologoustissue specific promoter segment operably linked to a nucleic acidsegment encoding the transcription factor, wherein the WRKY11 has atleast 95% sequence identity to SEQ ID NO:19, and wherein the plant hasat least 4% increased levels of secondary wall cellulose compared to awild type plant of the same species without the heterologous nucleicacid.
 10. A plant comprising a heterologous nucleic acid comprising asecondary wall cellulose synthase (CESA) promoter operably linked to anucleic acid segment encoding a WRKY11 plant transcription factor havingat least 95% sequence identity to SEQ NO:19, and wherein the plant hasat least 4% increased levels of secondary wall cellulose compared to awild type plant of the same species without the heterologous nucleicacid.
 11. The plant of claim 10, wherein the promoter is a secondarywall cellulose synthase 4 (CESA4) promoter, or a secondary wallcellulose synthase 7 (CESA7) promoter operably linked to a nucleic acidsegment encoding the WRKY11 plant transcription factor.